Selectively altering microbiota for immune modulation

ABSTRACT

The invention relates to methods of modulating immune cells in a patient by altering microbiota of the patient. The invention also relates to methods of modulating treatments or therapies in a subject organism by altering microbiota of the subject. The invention also relates to cell populations, systems, arrays, cells, RNA, kits and other means for effecting this. In an example, advantageously selective targeting of a particular species in a human gut microbiota using guided nucleic acid modification is carried out to effect the alteration.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Continuation Application of U.S. patentapplication Ser. No. 16/192,752 filed Nov. 15, 2018, which isContinuation Application of U.S. patent application Ser. No. 15/820,296filed on Nov. 21, 2017, which is a Continuation Application under 35U.S.C. § 120 of International Patent Application No. PCT/EP2017/063593filed on Jun. 4, 2017, which claims priority benefit to United KingdomPatent Application No. GB1609811.3 filed on Jun. 5, 2017, the contentsof which are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 786212000205SEQLIST.TXT,date recorded: Apr. 19, 2019, size: 8 KB).

FIELD OF THE INVENTION

The invention relates to methods of modulating immune cells in a patient(endogenous cells of the patient and/or administered cells, such as viaadoptive cell therapy) by altering microbiota of the patient. Theinvention also relates to methods of modulating treatments or therapiesin a subject organism by altering microbiota of the subject. Theinvention also relates cell populations, systems, kits and other meansfor effecting this. In an example, advantageously selective targeting ofa particular species in a human gut microbiota using guided nucleic acidmodification is carried out to effect the alteration.

BACKGROUND OF THE INVENTION

One approach to immunotherapy involves engineering patients' own (or adonor's) immune cells to express cell-surface antigen receptors (CARs)that recognise and attack tumours. Although this approach, calledadoptive cell transfer (ACT), has been restricted to small clinicaltrials so far, treatments using these engineered immune cells havegenerated some remarkable responses in patients with advanced cancer.

The Chimeric Antigen Receptor (CAR) consists of an antibody-derivedtargeting domain fused with T-cell signaling domains that, whenexpressed by a T-cell, endows the T-cell with antigen specificitydetermined by the targeting domain of the CAR. CARs can potentiallyredirect the effector functions of a T-cell towards any protein andnon-protein target expressed on the cell surface as long as anantibody-based targeting domain is available. This strategy therebyavoids the requirement of antigen processing and presentation by thetarget cell and is applicable to non-classical T-cell targets likecarbohydrates. This circumvention of HLA-restriction means that the CART-cell approach can be used as a generic tool broadening the potentialof applicability of adoptive T-cell therapy. See, eg, Methods Mol Biol.2012; 907:645-66. doi: 10.1007/978-1-61779-974-7_36, “Chimeric antigenreceptors for T-cell based therapy”, Cheadle E J et al.

The first CAR-T construct was described in a 1989 paper by immunotherapypioneer Zelig Eshhar in PNAS. The structure of the CAR now comprises atransmembrane polypeptide chain which is a chimaera of different domainsfrom different cellular proteins. For example, the CAR has anextracellular part joined (often by a linker and/or a hinge region) toan intracellular part, with a transmembrane portion of the CAR embeddingthe receptor in the membrane of an immune cell, normally a T-cell. Theextracellular moiety includes an antibody binding site (usually in theform of an scFv, such as derived from a mouse mAb) that recognizes atarget antigen, that commonly is a tumour associated antigen (TAA) onthe surface of cancer cells. Antigen recognition in this way dispenseswith the need to rely on TCRs that require MHC-restricted antigenpresentation, and where binding affinities may be relatively low. Theintracellular moiety of the CAR typically includes a CD3-zeta (CD3ζ)domain for intracellular signaling when antigen is bound to theextracellular binding site. Later generation CARs also include a furtherdomain that enhances T-cell mediated responses, which often is a 4-1BB(CD137) or CD28 intracellular domain. On encountering the cognateantigen ligand for the CAR binding site, the CAR can activateintracellular signaling and thus activation of the CAR T-cell to enhancetumour cell killing.

Most CAR-Ts expand in vivo so dose titration in a conventional sense isdifficult, and in many cases the engineered T-cells appear to be active“forever”—i.e., the observation of on-going B-cell aplasia seen in mostof the CD19 CAR-T clinical studies to date. This poses a serious problemfor CAR T-cell approaches. Some observed risks are discussed in DiscovMed. 2014 November; 18(100):265-71, “Challenges to chimeric antigenreceptor (CAR)-T cell therapy for cancer”, Magee M S & Snook A E, whichexplains that the first serious adverse event following CAR-T celltreatment occurred in a patient with colorectal cancer metastatic to thelung and liver (Morgan et al., 2010). This patient was treated with Tcells expressing a third-generation CAR targeting epidermal growthfactor receptor 2 (ERBB2, HER2). The CAR contained an scFv derived fromthe 4D5 antibody (trastuzumab) that is FDA approved for the treatment ofHER2-positive breast cancers (Zhao et al., 2009). The patient developedrespiratory distress within 15 minutes of receiving a single dose of1010 CAR-T cells, followed by multiple cardiac arrests over the courseof 5 days, eventually leading to death. Serum analysis four hours aftertreatment revealed marked increases in the cytokines IFNγ, GM-CSF, TNFα,IL-6, and IL-10. CAR-T cells were found in the lung and abdominal andmediastinal lymph nodes, but not in tumour metastases. The investigatorsattributed toxicity to recognition of HER2 in lung epithelium resultingin inflammatory cytokine release producing pulmonary toxicity andcytokine release syndrome (CRS) causing multi-organ failure (Morgan etal., 2010). Trials utilizing second-generation HER2-targeted CARsderived from a different antibody (FRP5) following conservativedose-escalation strategies are currently underway for a variety of HER2+malignancies by other investigators (clinicaltrials.gov identifiersNCT01109095, NCT00889954, and NCT00902044).

A variation on the CAR T-cell theme are antibody-coupled T-cell receptor(ACTR) therapeutics, which use CD16A (FCγRIIIA) to bind to Fc regions oftumour-specific IgG (see eg, WO2015/058018, U52015139943). The aim is toenable more control of CAR T-cell activity in vivo by titrating IgGadministered to patients. The CD16 binding sites of the CAR-T-cells maybe free, however, to also bind to endogenous IgG of the patients andthis reduces the attractiveness of the approach. The approach also needsto consider the inherently long half-life of IgG in the body (around 20days for IgG in man), which may limit control of CAR-cell activity.Ongoing studies may assess the risk of this.

It would be desirable to provide an alternative way to modulate(downregulate or upregulate) immune cell-based therapies, likeCAR-T-cell approaches and other cell-based approaches. It would also bedesirable to provide a way to address diseases and conditions mediatedby endogenous immune cells, such as autoimmune, inflammatory andinfectious diseases and conditions.

STATEMENTS OF INVENTION

The invention provides guided nucleases, host cell modifying(HM)-CRISPR/Cas systems, gRNAs, HM-arrays, HM-crRNA, HM-Cas, HM-TALENs,HM-meganucleases, HM-zinc fingers and methods as set out in the claimsherein.

Medical practice often involves the administration of antibiotics topatients. Such treatments can typically involve administration ofbroad-spectrum antibiotics, or antibiotics that target manygram-positive bacterial species or many gram-negative species withoutdiscrimination. Similarly, use of broad-spectrum antibiotics in farmingand agriculture, for example, raise environmental concerns, includingentry of such antibiotics into the human and animal food chain which maybe deleterious to health and may add to development of microbialresistance. Rather, the invention involves selective targeting of afirst microbiota species or strain. As shown in the worked examplesherein, selective targeting of a particular bacterial species has beenachieved using guided nuclease targeting of the genome of the selectedspecies, whilst at the same time sparing phylogenetically relatedspecies and strains. Furthermore, the invention realises the role thatmicrobiota bacteria and archaea play in shaping immune function inhumans and animals, as discussed further below.

Thus, the invention relates to methods of modulating immune cells in apatient (endogenous cells of the patient and/or administered cells, suchas via adoptive cell therapy) by altering microbiota of the patient. Inan example, advantageously selective targeting of a species in amicrobiota (eg, gut microbiota) is carried out to effect the alteration.Selective targeting may, for example, avoid targeting of related speciesor strains, such as species of the same phylum or such as a differentstrain of the same species.

For example, the invention provides for modulating immune cell-based orother therapy of diseases and conditions in patients and subjects byaltering microbiota, as well as systems, kits and other means foreffecting this.

For example, the invention provides for treating or reducing diseasesand conditions in patients by altering microbiota, wherein the diseasesand conditions are those mediated by immune cells (eg, T-cells) oraddressed by altering immune cell activities or populations in patients.Embodiments are cancers, autoimmune diseases or conditions, inflammatorydiseases or conditions, viral infections (eg, HIV infection of humanpatients), or diseases or conditions mediated or caused by viralinfections.

The invention also relates to methods of modulating treatments ortherapies in a subject organism (eg, a plant, yeast, human or animalpatient) by altering microbiota of the subject. Examples of therapiesare adoptive cell therapy, antibody therapy (eg, immune checkpointinhibition), radiation therapy, chemotherapy, eg, for treatment orprevention of a disease or condition in a patient.

In a first configuration the invention provides

A method of modulating a therapy of a disease or condition in a patient,the method comprising

a. Carrying out the therapy in the patient; and

b. Causing gut bacterial microbiota dysbiosis in the patient, wherebysaid dysbiosis modulates the therapy in the patient by modulating immunecells in the patient.

In another aspect, the first configuration the invention provides

A method of modulating a therapy of a disease or condition in a human oranimal patient, the method comprising

a. Carrying out the therapy in the patient; and

b. Causing bacterial (eg, gut bacterial) microbiota dysbiosis in thepatient, whereby said dysbiosis modulates the therapy in the patient bymodulating immune cells in the patient;

wherein the therapy comprises adoptive immune cell therapy (eg, adoptiveT-cell therapy, eg, CAR-T cell administration to the patient).

In another aspect, the first configuration the invention provides

A method of modulating a therapy of a disease or condition in a human oranimal patient, the method comprising

a. Carrying out the therapy in the patient; and

b. Causing bacterial (eg, gut bacterial) microbiota dysbiosis in thepatient, whereby said dysbiosis modulates the therapy in the patient;

wherein the therapy comprises administering an immune checkpointinhibitor (eg, an anti-PD-L1, anti-PD-1, anti-CTLA4 or anti-TIM3inhibitor, eg, an antibody) to the patient.

In another aspect, the first configuration the invention provides

A method of modulating a therapy of a disease or condition in a human oranimal patient, the method comprising

a. Carrying out the therapy in the patient; and

b. Causing bacterial (eg, gut bacterial) microbiota dysbiosis in thepatient, whereby said dysbiosis modulates the therapy in the patient;

wherein the therapy comprises administering an antibody (eg, ananti-PD-L1, anti-PD-1, anti-CTLA4 or anti-TIM3 antibody; or an anti-TNFasuperfamily member antibody, eg, an anti-TNFa, TNFR1 or BAFF antibody;or, an anti-IL6R or anti-IL-4Ra antibody; or an anti-PCSK9 antibody) tothe patient.

In another aspect, the first configuration the invention provides

A method of modulating a treatment in a subject, the method comprising

a. Carrying out the treatment in the subject; and

b. Causing microbiota dysbiosis in the subject, whereby said dysbiosismodulates the treatment in the subject.

In an example, the subject or patient is a human. In an example, thesubject or patient is a non-human animal. In an example, the subject isa plant, and optionally the treatment is a plant growth-promotingtreatment, growth-inhibiting treatment, pesticide treatment, nitrogenfixing promotion treatment, herbicidal treatment or fertilizertreatment. In an example, the subject is a yeast, and optionally thetreatment is a yeast growth-promoting treatment or growth-inhibitingtreatment.

In an example, the modulating augments, upregulates, downregulates,inhibits, enhances or potentiates the treatment or therapy of thesubject or patient. In an example, the treatment or therapy is effectivein the subject or patient, wherein the treatment or therapy is noteffective or has reduced or increased efficacy in the subject, patientor a control subject or patient that has not been subject to themodulation. The control is of the same species as the subject orpatient, and optionally the same age and/or sex. In an example,bacterial or archaeal host cells are killed or growth thereof isinhibited in the subject or patient using a method of an invention,wherein the control comprises cells of the same bacterial or archaealspecies and the cells are not killed or growth inhibited by a method ofthe inventon.

In an example, steps (a) and (b) are carried out simultaneously. In anexample, step (a) is carried out before step (b). In an example, step(b) is carried out before step (a), and optionally step (b) is performedagain after (a).

In an embodiment, the invention provides

A method of modulating a treatment in a plant or yeast, the methodcomprising

a. Carrying out the treatment in the plant or yeast; and

b. Causing bacterial microbiota dysbiosis in the plant or yeast, wherebysaid dysbiosis modulates the treatment in the subject;

wherein the treatment is a growth-promoting treatment, growth-inhibitingtreatment, pesticide treatment, nitrogen fixing promotion treatment,herbicidal treatment or fertilizer treatment.

Causing microbial dysbiosis in the subject, patient, plant or yeast is,in an example comprises causing microbial dysbiosis on a surface of thesubject, patient, plant or yeast, eg, on a leaf surface (when thesubject is a plant) or on skin, lung, ocular or mucosal surface (whenthe subject or patient is a human or animal).

Instead of or additionally to causing bacterial dysbiosis, the inventioncomprises in step (b) causing archaeal microbiota dysbiosis in saidsubject, patient, plant or yeast.

For example, the disease or condition is an autoimmune disease orcondition (eg, SLE) and the therapy is a treatment therefor, eg,administration of a tumor necrosis factor ligand superfamily memberantagonist, eg, an anti-B-cell activating factor (BAFF) antibody, suchas BENLYSTA® (belimumab) or a generic version thereof. For example, thedisease or condition is an inflammatory disease or condition (eg,rheumatoid arthritis, IBD, Crohn's disease, colitis or psoriasis) andthe therapy is a treatment therefor, eg, administration of sarilumab,dupilumab, a tumor necrosis factor ligand superfamily member antagonist,eg, an anti-TNF alpha antibody or trap, such as HUMIRA® (adalimumab),REMICADE® (infliximab), SIMPONI® (golimumab) or ENBREL® (etanercept) ora generic version thereof. For example, the disease or condition is aviral infection or mediated by a viral infection (eg, HIV infection) andthe therapy is a treatment therefor, eg, administration of ananti-retroviral medicament or an anti-HIV vaccine. For example, thedisease or condition is a cancer (eg, melanoma, NSCLC, breast cancer orpancreatic cancer) and the therapy is a treatment therefor, eg,administration of a chemotherapeutic agent, eg, a checkpoint inhibitoror agonist antibody such as an anti-CTLA4, PD-1, PD-L1, PD-L2, LAG3,OX40, CD28, BTLA, CD137, CD27, HVEM, KIR, TIM-3, VISTA, ICOS, GITR,TIGIT or SIRPa antibody. In an example, the antibody is a bispecificantibody that specifically binds first and second targets selected fromCTLA4, PD-1, PD-L1, PD-L2, LAG3, OX40, CD28, BTLA, CD137, CD27, HVEM,KIR, TIM-3, VISTA, ICOS, GITR, TIGIT and SIRPa, eg, wherein the firsttarget is CTLA4 and the second target is LAG3 or PD-1. Optionally, theantibody is a human gamma-1 antibody and/or may be enhanced for ADCC orCDC. For example, the therapy is a vaccine therapy, eg, a cancer vaccinetherapy or a vaccine therapy for treating or preventing an infection orinfectious disease, such as malaria, HIV infection, tuberculosisinfection, cholera, Salmonella typhimurium infection, C dificileinfection, Bordetella pertussis infection or chlamydia infection.

An embodiment of the first configuration provides

A method of modulating a cell therapy of a disease or condition in apatient, the method comprising

-   -   a. Carrying out cell therapy in the patient, comprising        administering a population of cells to the patient, wherein        administration of said cells is capable of treating the disease        or condition in the patient; and    -   b. Causing gut bacterial microbiota dysbiosis in the patient,        whereby said dysbiosis modulates the cell therapy in the        patient.

In an example the cell therapy is an adoptive immune cell therapy, suchas CAR-T or TILs therapy for the treatment of a cancer.

In a second configuration the invention provides

A method of treating or reducing the risk of a disease or condition in apatient, wherein the disease or condition is mediated by immune cells(eg, T-cells) in the patient, the method comprising causing gutbacterial microbiota dysbiosis in the patient, whereby said dysbiosismodulates immune cells (eg, T_(H)17 cells) in the patient, therebytreating or reducing the risk of said disease or condition in thepatient.

For example, the disease or condition is an autoimmune disease orcondition (eg, SLE), an inflammatory disease or condition (eg,rheumatoid arthritis, IBD, Crohn's disease, colitis or psoriasis), aviral infection or mediated by a viral infection (eg, HIV infection).

In an example, microbiota dysbiosis is effected by killing one or moretarget bacterial species in the microbiota or inhibiting growth of apopulation of said bacteria in the microbiota. In an example, microbiotadysbiosis is effected by killing one or more target archaeal species inthe microbiota or inhibiting growth of a population of said archaea inthe microbiota.

In a third configuration the invention provides

A method of modulating an adoptive immune cell therapy of a disease orcondition in a patient, the method comprising

-   -   a. Carrying out adoptive immune cell therapy in the patient,        comprising administering a population of immune cells to the        patient, wherein administration of said immune cells is capable        of treating the disease or condition in the patient; and    -   b. Altering the relative proportion of a sub-population of cells        of a first bacterial species or strain, or archaeal species or        strain, in a microbiota (eg, gut microbiota) of the patient,        thereby producing an altered microbiota that modulates the        immune cell therapy in the patient.

In another aspect, the third configuration the invention provides

A method of modulating a therapy of a disease or condition in a human oranimal patient, the method comprising

a. Carrying out the therapy in the patient; and

b. Altering the relative proportion of a sub-population of cells of afirst bacterial species or strain, or archaeal species or strain, in amicrobiota (eg, gut microbiota) of the patient, thereby producing analtered microbiota that modulates the therapy in the patient;wherein the therapy comprises administering an immune checkpointinhibitor (eg, an anti-PD-L1, anti-PD-1, anti-CTLA4 or anti-TIM3inhibitor, eg, an antibody) to the patient.

In another aspect, the third configuration the invention provides

A method of modulating a therapy of a disease or condition in a human oranimal patient, the method comprising

a. Carrying out the therapy in the patient; and

b. Altering the relative proportion of a sub-population of cells of afirst bacterial species or strain, or archaeal species or strain, in amicrobiota (eg, gut microbiota) of the patient, thereby producing analtered microbiota that modulates the therapy in the patient;wherein the therapy comprises administering an antibody (eg, ananti-PD-L1, anti-PD-1, anti-CTLA4 or anti-TIM3 antibody; or an anti-TNFasuperfamily member antibody, eg, an anti-TNFa, TNFR1 or BAFF antibody;or, an anti-IL6R or anti-IL-4Ra antibody; or an anti-PCSK9 antibody) tothe patient.

In another aspect, the third configuration the invention provides

A method of modulating a treatment in a subject, the method comprising

a. Carrying out the treatment in the subject; and

b. Altering the relative proportion of a sub-population of cells of afirst bacterial species or strain, or archaeal species or strain, in amicrobiota of the subject, whereby said dysbiosis modulates thetreatment in the subject.

In an example, the subject or patient is a human. In an example, thesubject or patient is a non-human animal. In an example, the subject isa plant, and optionally the treatment is a plant growth-promotingtreatment, growth-inhibiting treatment, pesticide treatment, nitrogenfixing promotion treatment, herbicidal treatment or fertilizertreatment. In an example, the subject is a yeast, and optionally thetreatment is a yeast growth-promoting treatment or growth-inhibitingtreatment.

In an example, the modulating augments, upregulates, downregulates,inhibits, enhances or potentiates the treatment or therapy of thesubject or patient. In an example, the treatment or therapy is effectivein the subject or patient, wherein the treatment or therapy is noteffective or has reduced or increased efficacy in the subject, patientor a control subject or patient that has not been subject to themodulation. The control is of the same species as the subject orpatient, and optionally the same age and/or sex. In an example,bacterial or archaeal host cells are killed or growth thereof isinhibited in the subject or patient using a method of an invention,wherein the control comprises cells of the same bacterial or archaealspecies and the cells are not killed or growth inhibited by a method ofthe invention.

In an example, steps (a) and (b) are carried out simultaneously. In anexample, step (a) is carried out before step (b). In an example, step(b) is carried out before step (a), and optionally step (b) is performedagain after (a).

In an embodiment, the invention provides

A method of modulating a treatment in a plant or yeast, the methodcomprising

a. Carrying out the treatment in the plant or yeast; and

b. Altering the relative proportion of a sub-population of cells of afirst bacterial species or strain, or archaeal species or strain, in amicrobiota of the plant or yeast, whereby said dysbiosis modulates thetreatment in the plant or yeast;

wherein the treatment is a growth-promoting treatment, growth-inhibitingtreatment, pesticide treatment, nitrogen fixing promotion treatment,herbicidal treatment or fertilizer treatment.

Said altering of the relative proportion of sub-population of cells inthe subject, patient, plant or yeast is, in an example comprises causingmicrobial dysbiosis on a surface of the subject, patient, plant oryeast, eg, on a leaf surface (when the subject is a plant) or on skin,lung, ocular or mucosal surface (when the subject or patient is a humanor animal).

The proportion of the first bacteria or archaea sub-population isincreased or decreased. In an example, the relative ratio of first andsecond bacterial species or strains is altered (eg, increased ordecreased); or the relative ratio of first and second archaeal speciesor strains is altered (eg, increased or decreased).

In an example, the adoptive immune cell therapy is CAR-T therapy for thetreatment of a cancer. In an example, the adoptive immune cell therapyis a TILs therapy for the treatment of a cancer.

In an example of the first or third configuration, the cells of step (a)are of a first type selected from the group consisting of CD4⁺ T-cells,CD8⁺ T-cells, T_(H)1 cells or T_(H)17 cells and step (b) upregulatescells of that type in the patient. This is useful for enhancing the cellbased therapy. In another example the cells of step (a) are of a firsttype selected from the group consisting of CD4⁺ T-cells, CD8⁺ T-cells,T_(H)1 cells or T_(H)17 cells and step (b) downregulates cells of thattype in the patient. This is useful for dampening down the cell basedtherapy or a side effect thereof (eg, CRS).

In an embodiment, the disbyosis or step (b) is carried out usingselective targeting of a bacterial or archaeal microbiota sub-populationusing CRISPR/Cas targeting of microbiota (eg, gut microbiota) bacteriaand/or archaea. In an example, the method comprises using guidednuclease (eg RNA-guided nuclease) cutting of a respective targetsequence in host cells to modify the target sequences, whereby hostcells are killed or the host cell population growth is reduced, therebyreducing the proportion of said sub-population in the microbiota.Suitable systems for carrying out the guided nuclease cutting are, forexample, engineered CRISPR/Cas systems, TALENs, meganucleases and zincfinger systems.

To this end, the inventors believe that they have demonstrated for thefirst time inhibition of population growth of a specific bacterialstrain in a mixed consortium of bacteria that naturally occur togetherin gut microbiota with one or more of the following features:—

-   -   Population growth inhibition using an engineered CRISPR/Cas        system by    -   targeting wild-type cells;    -   harnessing of wild-type endogenous Cas nuclease activity;    -   targeting essential and antibiotic resistance genes;    -   wherein the targets are wild-type sequences.

The inventors have demonstrated this in a mixed population of human gutmicrobiota bacteria with the following features:—

-   -   targeting bacterial growth inhibition in a mixed population of        human gut microbiota species;    -   wherein the population comprises three different species;    -   comprising selective killing of one of those species and sparing        cells of the other species;    -   targeting cell growth inhibition in the presence of a        phylogenetically-close other human gut microbiota species, which        is spared such inhbition;    -   targeting cell growth inhibition in a mixed population of human        gut microbiota bacteria comprising target Firmicutes species and        non-Firmicutes species;    -   targeting cell growth inhibition of a specific Firmicutes        species whilst sparing a different Firmicutes species in a mixed        population of human gut microbiota bacteria;    -   targeting cell growth inhibition of a specific gram positive        bacterial strain whilst sparing a different gram positive        bacterial species in a mixed population of human gut microbiota        bacteria;    -   targeting a human gut microbiota bacterial species whilst        sparing a commensul human gut bacterial species;    -   targeting a human gut microbiota bacterial species whilst        sparing a priobiotic human gut bacterial species;    -   targeting cell growth inhibition in a mixed population of human        gut microbiota bacteria on a surface;    -   achieving at least a 10-fold growth inhibition of a specific        bacterial species alone or when mixed with a plurality of other        bacterial species in a consortium of human gut microbiota        bacteria; and    -   achieving at least a 10-fold growth inhibition of two different        strains of a specific human gut microbiota bacterial species.

The invention provides:

An ex vivo population of immune cells for use in a method of adoptivecell therapy of a patient for treating or preventing a disease orcondition in the patient, the method comprising

-   -   a. Carrying out adoptive immune cell therapy in the patient,        comprising administering cells of said population to the        patient, wherein administration of said immune cells is capable        of treating the disease or condition in the patient; and    -   b. Causing gut bacterial microbiota dysbiosis in the patient,        whereby said dysbiosis modulates the immune cell therapy in the        patient and said disease or condition is treated or prevented.

The invention provides

An ex vivo population of immune cells for use in a method of adoptivecell therapy of a patient for treating or preventing a disease orcondition in the patient, the method comprising

-   -   a. Carrying out adoptive immune cell therapy in the patient,        comprising administering cells of said population to the        patient, wherein administration of said immune cells is capable        of treating the disease or condition in the patient; and    -   b. Altering the relative proportion of a sub-population of cells        of a first bacterial species or strain, or archaeal species or        strain, in the gut microbiota of the patient, thereby producing        an altered gut microbiota that modulates the immune cell therapy        in the patient. The invention also provides CRISPR/Cas systems,        arrays, cRNAs and kits for carrying out a method of the        invention.

The invention also relates to systems, kits and other means foreffecting the method.

Any features on one configuration herein are, in an example, combinedwith a different configuration of the invention for possible inclusionof such combination in one or more claims herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows s Xylose inducible system.

FIG. 2 shows a ST1-CRISPR array.

FIG. 3 shows a spot assay on TH-agar of the strains used in this work.All strains were grown on TH-agar at 37° C. for 20 hours. Serialdilutions of overnight cultures were done in duplicate for E. coli, LLactis and S. mutans, and triplicate for both strains of S. thermophilusin order to count individual colonies.

FIG. 4 shows selective growth of S. thermophilus, S. mutans, L. lactisand E. coli under different culture conditions. Tetracycline cannot beused to selectively grown S. thermophilus LMD-9. However, 3 g 1⁻¹ of PEAproved to selectively grow S. thermophilus LMD-9 while limiting growthof E. coli.

FIG. 5 illustrates construction of two xylose induction cassettes.

FIG. 6 demonstrated characterization of the xylose inducible cassette inStreptoccocus thermophilus LMD-9 with the plasmidpBAV1KT5-XylR-mCherry-Pldha. A clear response in fluorescence can beobserved with increasing amount of xylose.

FIG. 7 illustrates the design of CRISPR array inpBAV1KT5-XylR-mCherry-P_(Idha+xylA). The array contains 2 spacersequences that target S. thermophilus genes under an inducible xylosepromoter and a tracrRNA under a strong constitutive promoter P_(3A).

FIG. 8 shows transformation efficiency of Streptoccocus thermophilusLMD-9 with the plasmid pBAV1KT5-XylR-CRISPR-P_(Idh+XylA) and withpBAV1KT5-XylR-CRISPR-P_(xylA).

FIG. 9 shows a schematic of the xylose-inducible CRISPR device. Uponinduction of xylose the CRISPR array targeting both polIII and tetA onthe S. thermophiles LMD-9 genome are expressed. Together with theconstitutively expressed tracrRNA a complex is formed with Cas9. Thiscomplex will introduce a double stranded break in the tetA and polIIIgenes in the S. thermophilus LMD-9 genome resulting in limited cellviability.

FIG. 10 shows growth inhibition of Streptoccocus thermophilus DSM20617(T) with the plasmid pBAV1KT5-XylR-CRISPR-PXylA orpBAV1KT5-XylR—CRISPR-Pldha+XylA, not induced and induced. Picture takenafter 63H of incubation. Colony counts in bottom left corner (toprow: >1000, >1000, bottom row: 336, 113).

FIG. 11 shows a maximum-likelihood phylogenetic tree of 16S sequencesfrom S. thermophilus, L. lactis and E. coli.

FIG. 12 shows the selective S thermophilus growth inhibition in aco-culture of E. coli, L. lactis and S. thermophiles harboring eitherthe pBAV1KT5-XylR-CRISPR-PxylA or the pBAV1KT5-XylR-CRISPR-PldhA+XylAplasmid. No growth difference is observed between E. coli harboring thepBAV1KT5-XylR-CRISPR-PxylA or the pBAV1KT5-XylR-CRISPR-PldhA+XylAplasmid. However, S. thermophiles (selectively grown on TH agarsupplemented with 2.5 gl-1 PEA) shows a decrease in transformationefficiency between the pBAV1KT5-XylR-CRISPR-PxylA (strong) or thepBAV1KT5-XylR-CRISPR-PldhA+XylA (weak) plasmid as we expected. We thusdemonstrated a selective growth inhibition of the target S. thermophilussub-population in the mixed population of cells. Colony counts in bottomleft corner (top row: >1000, >1000, 68, bottom row: >1000, >1000, 32).

FIG. 13 shows regulators controlling the expression of spCas9 and theself-targeting sgRNA targeting the ribosomal RNA subunit 16s.

FIG. 14 shows specific targeting of E. coli strain by an exogenousCRISPR-Cas system. The sgRNA target the genome of K-12 derived E. colistrains, like E. coli TOP10, while the other strain tested wasunaffected.

FIG. 15 shows spot assay with serial dilutions of individual bacterialspecies used in this study and mixed culture in TH agar withoutinduction of CRISPR-Cas9 system.

FIG. 16 shows spot assay of the dilution 10³ on different selectivemedia. TH with 2.5 g 1⁻¹ PEA is a selective media for B. subtilis alone.MacConkey supplemented with maltose is a selective and differentialculture medium for bacteria designed to selectively isolateGram-negative and enteric bacilli and differentiate them based onmaltose fermentation. Therefore TOP10 ΔmalK mutant makes white colonieson the plates while Nissle makes pink colonies; A is E coli ΔmalK, B isE coli Nissile, C is B subtilis, D is L lactis, E is mixed culture; theimages at MacConkey−/B and E appear pink; the images at MacConkey+/B andE appear pink.

FIG. 17 shows selective growth of the bacteria used in this study ondifferent media and selective plates.

DETAILED DESCRIPTION

In the worked Example below, growth inhibition was addressed in a mixedpopulation of human gut microbiota bacterial species. A >10-foldpopulation growth inhibition in a selectively targeted species (a grampositive Firmicutes population) was achieved, sparing non-targetedcommensal bacteria in the consortium. The inventors have realised theuseful application of this for altering microbiota, such as gutmicrobiota, in situ in patients, thereby enabling immune cell modulationin the patient in response to the altered microbiota. The inventors alsorealised application to modulating treatments in subjects such as plantsand yeast that comprise microbiota that can be altered. The inventorsfurthermore realised the utility for modulating immune cell-basedtherapies in patients or for treating or preventing immune cell-mediateddiseases or conditions in patients, such as autoimmune diseases,inflammatory diseases and viral infections (eg, HIV infection ofhumans). The inventors realised the utility of causing dysbiosis of gut,skin, vaginal, nasal, ocular, lung, GI tract, rectal, scrotal, ear, skinor hair microbiota for effecting such modulation in a human or animalsubject or patient.

As used herein “dysbiosis” refers to a change of the bacterial and/orarchaeal balance of the microbiota, eg, gut microbiota. Change isrelative to the balance prior to (eg, immediately prior or no more thana day before) carrying out the method. The change can be one or more of(i) an increase in the proportion of a first species (bacterial orarchaeal species) or strain in the microbiota (eg, gut microbiota) (eg,B fragalis or thetaiotamicron); (ii) an increase in the relativeproportion of first and second species (eg, B fragalis versus Cdificile; or S thermophilus v E coli or L lactis), first and secondstrains of the same species, or first and second phyla which aredifferent from each other (eg, Bacteriodetes versus Firmicutes); (iii)an addition of a species or strain that was not comprised by themicrobiota prior to the treatment method; (iv) a decrease in theproportion of a first species (bacterial or archaeal species) or strainin the microbiota (eg, C dificile or S thermophilus); (v) a decrease inthe relative proportion of first and second species (eg, B fragalisversus C dificile; or S thermophilus v E coli or L lactis), first andsecond strains of the same species, or first and second phyla which aredifferent from each other (eg, Bacteriodetes versus Firmicutes); and(vi) a removal of a species or strain that was not comprised by themicrobiota prior to the treatment method. Dysbiosis may be effected, forexample, using one or more selective antibacterial agents (eg,CRISPR-based or other guided nucleases described herein) of byadministering one or more bacterial and/or archaeal transplants to thepatient or subject to alter the balance of the microbiota, eg, gutmicrobiota.

The impact of the immune system on microbiota composition is suggestedby several immune deficiencies that alter microbial communities in waysthat predispose to disease. For example, Garrett et al. studied micethat lack the transcription factor T-bet (encoded by Tbx21), whichgoverns inflammatory responses in cells of both the innate and theadaptive immune system (Cell. 2007 Oct. 5; 131(1):33-45, “Communicableulcerative colitis induced by T-bet deficiency in the innate immunesystem”, Garrett W S et al.). When Tbx21−/−mice were crossed ontoRag2−/−mice, which lack adaptive immunity, the Tbx21−/−/Rag2−/−progenydeveloped ulcerative colitis in a microbiota-dependent mannerRemarkably, this colitis phenotype was transmissible to wild-type miceby adoptive transfer of the Tbx21−/−/Rag2−/−microbiota. Thisdemonstrated that altered microbiota were sufficient to induce disease.Another example of immune-driven dysbiosis is seen in mice deficient forepithelial cell expression of the inflammasome component NLRP6. Thesemice develop an altered microbiota with increased abundance of membersof the Bacteroidetes phylum associated with increased intestinalinflammatory cell recruitment and susceptibility to chemically-inducedcolitis.

It has become evident that individual commensal species influence themakeup of lamina propria T lymphocyte subsets that have distincteffector functions. Homeostasis in the gut mucosa is maintained by asystem of checks and balances between potentially pro-inflammatorycells, which include T_(H)1 cells that produce interferon-γ, T_(H)17cells that produce IL-17a, IL-17f, and IL-22, diverse innate lymphoidcells with cytokine effector features resembling T_(H)2 andT_(H)17cells, and anti-inflammatory Foxp3⁺ regulatory T cells(L_(regs)).

A particular application of the invention is found in the shaping ofT_(H)17 cell populations in patients. Such cells have been implicated inautoimmune and inflammatory disorders. These cells were described in:Harrington L E, Hatton R D, Mangan P R, et al., “Interleukin17-producing CD41 effector T cells develop via a lineage distinct fromthe T helper type 1 and 2 lineages”, Nat Immunol. 2005; 6(11):1123-1132; and Park H, Li Z, Yang X O, et al., “A distinct lineage ofCD4 T cells regulates tissue inflammation by producing interleukin 17”,Nat Immunol. 2005; 6(11):1133-1141. In the case of autoimmune disorders,T_(H)17 cell over activation can cause an inappropriate amount ofinflammation, like in the case of multiple sclerosis, rheumatoidarthritis, and psoriasis. T_(H)17cells have also been shown to benecessary for maintenance of mucosal immunity. T_(H)17cells maycontribute to the development of late phase asthmatic response due toincreases in gene expression relative to T_(reg) cells.

In HIV, the loss of T_(H)17 cell populations can contribute to chronicinfection. The depletion of T_(H)17 cell populations in the intestinedisrupts the intestinal barrier, increases levels of movement ofbacteria out of the gut through microbial translocation, and contributesto chronic HIV infection and progression to AIDS. Microbialtranslocation results in bacteria moving from out of the gut lumen, intothe lamina propia, to the lymph nodes, and beyond into non-lymphatictissues. It can cause the constant immune activation seen through thebody in the late stages of HIV. Increasing T_(H)17 cell populations inthe intestine has been shown to be both an effective treatment as wellas possibly preventative. Although all CD4⁺ T cells gut are severelydepleted by HIV, the loss of intestinal T_(H)17 cells in particular hasbeen linked to symptoms of chronic, pathogenic HIV and SIV infection.Microbial translocation is a major factor that contributes to chronicinflammation and immune activation in the context of HIV. Innon-pathogenic cases of SIV, microbial translocation is not observed.T_(H)17 cells prevent severe HIV infection by maintaining the intestinalepithelial barrier during HIV infection in the gut. Because of theirhigh levels of CCR5 expression, the coreceptor for HIV, they arepreferentially infected and depleted. Thus, it is through T_(H)17 celldepletion that microbial translocation occurs. Additionally, the loss ofT_(H)17 cells in the intestine leads to a loss of balance betweeninflammatory T_(H)17 cells and T_(reg) cells, their anti-inflammatorycounterparts. Because of their immunosuppressive properties, they arethought to decrease the anti-viral response to HIV, contributing topathogenesis. There is more T_(reg) activity compared to T_(H)17activity, and the immune response to the virus is less aggressive andeffective. Revitalizing T_(H)17 cells has been shown to decreasesymptoms of chronic infection, including decreased inflammation, andresults in improved responses to highly active anti-retroviral treatment(HAART). This is an important finding—microbial translocation generallyresults in unresponsiveness to HAART. Patients continue to exhibitsymptoms and do not show as reduced a viral load as expected. In anSIV-rhesus monkey model, It was found that administering IL-21, acytokine shown to encourage T_(H)17 differentiation and proliferation,decreases microbial translocation by increasing T_(H)17 cellpopulations.

In an example of the method, IL-21, IL-15 and/or IL-2 is administered tothe patient sequentially or simultaneously with the cell population.This is useful for further modulating immune cell populations in thepatient.

Yang et al. observed that the presence of T_(H)17 cells in mice requirescolonisation of mice with microbiota. Segmented filamentous bacteria(SFB) were sufficient to induce T_(H)17 cells and promoteT_(H)17-dependent autoimmune disease in animal models (Nature, 2014 Jun.5; 510(7503):152-6. doi: 10.1038/nature13279. Epub 2014 Apr. 13,“Focused specificity of intestinal Th17 cells towards commensalbacterial antigens”, Yang Y et al.). SFB appear able to penetrate themucus layer overlying the intestinal epithelial cells in the terminalileum, and they interact closely with the epithelial cells, inducinghost cell actin polymerization at the site of interaction and,presumably, signaling events that result in a T_(H)17 polarizingenvironment within the lamina propria.

In an example, the first bacteria are of a species or strain comprisinga 16s rDNA sequence that is at least 80, 85, 90, 95, 96, 97, 98 or 99%identical to a 16s rDNA sequence of a segmented filamentous bacterium.In an embodiment, the method increases the proportion of the firstbacteria, wherein T_(H)17 cells in the patient are upregulated, eg,wherein the disease is a cancer or a viral infection (eg, HIV). In anembodiment, the method decreases the proportion of the first bacteria,wherein T_(H)17 cells in the patient are downregulated, eg, wherein thedisease or condition is an autoimmune or inflammatory disease orcondition, or for reducing the risk of CRS in a cancer patient receivingACT.

In an example, the method treats or prevents an allergic disease orcondition, eg, asthma. In an example, the method treats or prevents anIgE-mediated disease or condition, eg, asthma.

In an example, the method reduces autotoxicity in the patient mediatedby T_(H)2 cell cytokine release.

López et al. observed that intestinal dysbiosis, characterised by areduced Firmicutes/Bacteroidetes ratio, has been reported in systemiclupus erythematosus (SLE) patients. In their study, in vitro culturesrevealed that microbiota isolated from SLE patient stool samples (SLE-M)promoted lymphocyte activation and T_(H)17 differentiation from naïveCD4⁺ lymphocytes to a greater extent than healthy control microbiota.Enrichment of SLE-M with T_(reg)-inducing bacteria showed that a mixtureof two Clostridia strains significantly reduced the T_(H)17/T_(H)1balance, whereas Bifidobacterium bifidum supplementation prevented CD4⁺lymphocyte over-activation. Ex vivo analyses of patient samples showedenlarged T_(H)17 and Foxp3*IL-17⁺ populations, suggesting a possibleT_(reg)-T_(H)17 trans-differentiation. Moreover, analyses of faecalmicrobiota revealed a negative correlation between IL-17⁺ populationsand Firmicutes in healthy controls, whereas in SLE this phylumcorrelated directly with serum levels of IFNγ, a T_(H)1 cytokineslightly reduced in patients. (Sci Rep. 2016 Apr. 5; 6:24072. doi:10.1038/srep24072, “Th17 responses and natural IgM antibodies arerelated to gut microbiota composition in systemic lupus erythematosuspatients”, Lopez P et al.).

Other bacteria have been shown to enhance the anti-inflammatory branchesof the adaptive immune system by directing the differentiation ofT_(regs) or by inducing IL-10 expression. For example, colonisation ofgnotobiotic mice with a complex cocktail of 46 mouse Clostridialstrains, originally isolated from mouse faeces and belonging mainly tocluster IV and XIVa of the Clostridium genus, results in the expansionof lamina propria and systemic T_(regs).

Bacteroides fragilis polysaccharide-A (PSA) impacts the development ofsystemic T cell responses. Colonization of germ-free mice withPSA-producing B. fragilis results in higher numbers of circulating CD4⁺T cells as compared to mice colonized with B. fragilis lacking PSA.PSA-producing B. fragilis also elicits higher T_(H)1 cell frequencies inthe circulation. Together, these findings show that commensal bacteriahave a general impact on immunity that reaches well beyond mucosaltissues.

The decrease in F. prausnitzii found in IBD patients is of interestbecause this bacteria is butyrate-producing, and its oral administrationreduces the severity of TNBS-induced colitis in mice. In an example, thefirst species is a butyrate-producing bacterial species (eg, F.prausnitzii) and the proportion of the first species in the microbiotais reduced, wherein the method downregulates T-effector and/or T-helpercells in the patient, thereby treating or preventing said disease orcondition (eg, an autoimmune or inflammatory disease or condition orCRS).

Archaea have traditionally been divided into five phyla, namelyCrenarchaeota, Euryarchaeota, Korarchaeota, Nanoarchaeota andThaumarchaeota. Based on the increasing wealth of whole genome data(mainly from environmental isolates), the archaeal phylogeny has beenrevisited recently: the four groups Korarchaeota, Crenarchaeota,Thaumarchaeota and the newly proposed Aigarchaeota have been comprisedinto one superphylum (the so-called TACK-superphylum) to the exclusionof Euryarchaeota and Nanoarchaeota. The first species in the method ofthe invention can be any of the archaea mentioned in this paragraph.

T cells mature in the thymus, express TCR (T cell receptor), and canexpress either CD8 glycoprotein on their surface and are called CD⁸⁺ Tcells (cytotoxic) or CD4 glycoprotein and are then called CD4 cells(helper T cells). CD⁴⁺ cells differentiate into different subsets: T_(H)(T helper)1, T_(H) 2, T_(H)9, T_(H)17, T_(H)22, T_(reg) (regulatory Tcells) and T_(fh), (follicular helper T cells), which are characterizedby different cytokine profiles. These different CD4⁺ subsets play acritical role in the immune and effector response functions of T cells.All CD4⁺ T_(H) subsets are differentiated from naïve CD4⁺ T cells byspecific cytokines: T_(H) 1 by IL-12 and IFN-γ (pro-inflammatorycytokine, with multiple roles such as increase of TLR (Toll-likereceptor), induction of cytokine secretion or macrophage activation);T_(H)2 by IL-4; T_(reg) by IL-2 and TGF-beta. Each T_(H) subset releasesspecific cytokines that can have either pro- or anti-inflammatoryfunctions, survival or protective functions. For example, T_(H)1releases IFN-γ and TNF; T_(H)2 releases IL-4 (an important survivalfactor for B-type lymphocytes), IL-5 and IL-13; T_(H)9 produces IL-9;T_(reg) secretes IL-10 (a cytokine with an immunosuppressive function,maintaining expression of FOXP3 transcription factor needed forsuppressive function of T_(reg) on other cells) and TGF-β; T_(H)17produces IL-17 (a cytokine playing an important role in host defenseagainst bacteria, and fungi).

An embodiment of the invention finds application for modulating CAR-Tand other adoptive immune-cell therapies (such as adoptive TILstherapy). Several reports have demonstrated differential roles ofdifferent types of cytokines released by CD4+ subsets, an importantconsideration when assessing CAR-T and other immune cell-basedtherapies. T_(H) 1 and T_(H) 2 CD4⁺ T cell subset cytokines were shownto drive different types of cytotoxicity generated by second generationCD28-containing CAR-T. Short-term toxicity was observed with high levelsof T_(H)1 cytokines, while high doses of T_(H) 2 type cytokinesgenerated chronic autocytotoxicity in animals that received secondgeneration CD19-specific CAR-T. CAR-T cells engineered to deliverinducible IL-12 modulated tumor stroma to destroy cancer. IL-12 releaseby engineered CAR-T cells increased anti-cancer activity by recruitingmacrophages. IL-12 released by CAR-T also induced reprogramming ofsuppressive cells, reversing their inhibitory functions suggesting itsevaluation in clinical trials. The persistence of CAR-T therapy wasshown to be dependent on the number of CD4⁺ cells and the number ofcentral memory cells in the infused product. CD8⁺ clones isolated fromcentral memory T cells but not from CD8⁺ effector cells persistedlong-term in vivo during adoptive T cell transfer in a nonhuman primatemodel, indicating the importance of specific T cell subset functions foreffective adoptive immunotherapy. It has also been shown that thecombination of CD8⁺ subset with CD4⁺ subset significantly enhanced Tcell adoptive transfer. CD4⁺ cells were shown to support development ofCD8⁺ memory functions, demonstrating the importance of both subsets andcombinations in immunotherapy trials. Several preclinical modelsdemonstrated the advantage of different T cell subsets for effectiveCAR-T therapy: CD8⁺ CD45RA⁺ CCR7⁺ CAR-T cells with closest to theT-memory stem cells phenotype cells produced greater anti-tumor activityof CAR-T cells; both CD8⁺ and CD4⁺ subsets expressed synergisticanti-tumor CAR-T activities.

In an example, the administered cell population is a population of CAR-Tcells comprising a combination of a CD8⁺ CAR-T subset with CD4⁺ CAR-Tsubset.

In an example of the invention, the cell therapy is an adoptive T-celltherapy and optionally cells selected from the group consisting of CD4+T-cells, CD8+ T-cells, TH1 cells and TH17 cells are administered to thepatient. In an example, cell therapy is enhanced by the method of theinvention, eg, immune cell cytotoxicity of cancer cells is enhanced inthe patient, or treatment of the disease or condition is enhanced. In anexample, cell therapy is reduced by the method of the invention, eg,immune cell cytotoxicity of cancer cells is reduced in the patient, orthe risk of CRS is reduced (eg, in a cancer patient). Thus, in anembodiment the method reduces or prevents the risk of cytokine releasesyndrome (CRS) in the patient. In an embodiment the method reduces orprevents the risk of an unwanted side-effect of the cell therapy (eg, aCAR-T therapy side effect in a human patient, such as CRS).

In an example, the immune cell population comprises CAR-T cells and/orT-cells expressing engineered T-cell receptors (TCRs) and/or tumourinfiltrating lymphocytes (TILs, eg, Engineered TILs). WO2013063361,US9113616, US20130109053, US20160081314 and WO2016044745 (whosedisclosures are incorporated herein by reference) describe suitabletransgenic in vivo platforms for generating CARs and TCRs for use ingenerating cells for use in the present invention. The immune cellpopulation may comprise engineered autologous or allogeneic immune cells(transplant), eg, T-cells, NK cells and/or TILs, eg, wherein the cellsand patient are human A benefit of autologous cells is that themodulation of the endogenous system is likely to be tuned similarly tomodulation of the cell transplanted autologous cells. In an embodiment,the administered cells and patient are of the same species or strain,for example, human or rodent (eg, mouse), for example, HLA or MHCmatched donor transplant and recipient patient.

In an example, the T-cells are CD4⁺ T-cells or T_(H)17 cells. Forexample, the administered CAR-T cells comprise a chimaeric antigenreceptor comprising an ICOS intracellular domain and optionally thecells are T_(H)17 cells. In an embodiment, the administered T-cells areCD8⁺ CD45RA⁺ CCR7⁺ CAR-T cells.

Adoptive transfer experiments in mice indicate that T_(H)17 cells havehigher in vivo survival and self-renewal capacity than T_(H)1 polarizedcells. In an example, therefore, T_(H)17 cells are modulated in thepatient, eg, upregulated, eg, expanded in the patient, or downregulated.These may be endogenous T-cells of the patient and/or cells that havebeen administered to the patient or progeny thereof. In an embodiment,RORγt-expressing T_(H)17 cells are upregulated, eg, expanded in thepatient. In an embodiment expression of one or more T_(H)17-relatedgenes is increased, eg, one or more of Rorc, Il22 and Il26. In anembodiment expression of one or more T_(H)1-related genes is increased,eg, one or more of Ifng, Tnfa and Tbx21 (T-bet). In an embodiment, inthis case the disease or condition is a cancer.

In an example, T_(reg) cells are modulated in the patient, eg,upregulated, eg, expanded in the patient, or downregulated. These may beendogenous T-cells of the patient and/or cells that have beenadministered to the patient or progeny thereof. In an embodiment, inthis case the disease or condition is an autoimmune, inflammatory orinfectious disease or condition when the T_(reg) cells are upregulated.

In an example, CD4⁺ cells are modulated in the patient, eg, upregulated,eg, expanded in the patient, or downregulated. These may be endogenouscells of the patient and/or cells that have been administered to thepatient or progeny thereof.

In an example, CD8⁺ cells are modulated in the patient, eg, upregulated,eg, expanded in the patient, or downregulated. These may be endogenouscells of the patient and/or cells that have been administered to thepatient or progeny thereof.

In an example, tumour infiltrating lymphocytes (TILs) are modulated inthe patient, eg, upregulated, eg, expanded in the patient, ordownregulated. These may be endogenous cells of the patient and/or cellsthat have been administered to the patient or progeny thereof.

In an example, memory cells, such as one or more of central memory Tcells (T_(CM)), effector memory T cells (T_(EM)), stem cell memory cells(T_(SCM)) and effector cells (T_(eff)), are upregulated in themicrobiota or patient, optionally wherein the cells are comprised by theimmune cell population administered to the patient and/or are progenythereof. In an embodiment, the memory cells are CD45RO⁺ CD62L⁺ or CD25⁺CD45RA⁻ CD45RO⁺ CD127⁺.

Upregulation of a cell population may, for example, be an increase inthe population size or proportion of cells of that type (eg, species orstrain) in the microbiota or patient or subject and/or an increase inthe activity (eg, cytotoxicity, effector function or suppressorfunction) of cells of that type in the microbiota or patient or subject.Downregulation of a cell population may, for example, be an decrease inthe population size or proportion of cells of that type (eg, species orstrain) in the microbiota or patient or subject and/or a decrease in theactivity (eg, cytotoxicity, effector function or suppressor function) ofcells of that type in the microbiota or patient or subject.

In an example, the cell therapy population comprises CAR-T cells (ie,respectively T-cells engineered to surface-express chimaeric antigenreceptors (CARs). Alternatively, the cells are CAR-TIL or CAR-NK cells.A CAR comprises an extracellular receptor domain for binding to a targetantigen (eg, a tumour cell antigen), a transmembrane moiety and anintracellular moiety comprising one or more (eg, first and second)signalling domains for signalling in the immune cell (eg, T-cell).Examples of suitable intracellular domains are well known, eg, acombination of a CD3ζ domain and one or more of an ICOS, CD28, OX40 or4-1BB signalling domain, eg, a combination of an ICOS and CD28; or ICOSand 41-BB; CD28 and 41-BB signalling domain

Optionally, the cell population is comprised by a transplant that isadministered to the patient to treat or prevent a disease (eg, a cancer,autoimmune disease, transplant rejection or GvHD) or the cell ortransplant is for such use.

In an example, the patient is a human, eg, is a woman; or a man

In an example, the patient or human has undergone lymphodepletion beforeadministration of the immune cell (eg, CAR-T cell).

Techniques for producing CARs and CAR T-cells are known and routine inthe art, and these can be generally applied to producing cells for usein the invention (eg, see WO2012079000A1; U.S. Pat. Nos. 8,906,682,8,911,993, 8,916,381, 8,975,071, 9,101,584, 9,102,760, 9,102,761,9,328,156, 9,464,140, 9,481,728, 9,499,629, 9,518,123, 9,540,445,US20130287748, US20130288368, US20130309258, US20140106449,US20140370017, US20150050729, US20150093822, US20150099299,US20150118202, US20160130355, US20160159907, US20160194404,US20160208012; J Immunother. 2009 September; 32(7): 689-702, doi:10.1097/CJI. 0b013e3181ac6138, “Construction and Pre-clinical Evaluationof an Anti-CD19 Chimeric Antigen Receptor”, James N. Kochenderfer et al;also WO2014012001 and US20150290244 for general methods applicable tothe present invention). For example, use of electroporation, retroviralvectors or lentiviral vectors—as will be known by the skilledaddressee—can be used to introduce nucleotide sequences encodingelements of the CAR into T-cells, NK cells, TILs or other immune cellsto produce the CAR-cells. Cells isolated from the patient (autologouscell sample) or from another donor of the same species (allogeneicsample) can be used to provide ancestor cells that are geneticallyengineered to include the CAR-encoding sequences. Expansion of cells canbe used in the process, as known in the art. For example, afterengineering CAR-cells, the cell population can be massively expandedusing routine techniques to produce a transplant that is administered(eg, transfused) into the patient. The patient can be a human onnon-human animal Nucleotide sequences for one or more of the CARelements (eg, for one or more of the signalling domains) can be clonedor sequenced using a cell obtained from the patient or from anotherdonor.

For example, the CAR comprises a first intracellular signalling domain,which is a human CD3 domain and the cells administered to the patientare human cells comprising an endogenous nucleotide sequence encodingsaid human CD3ζ domain. In an example, the CD3 zeta signaling domaincomprises SEQ ID NO: 1, i.e., the amino acid sequence of SEQ ID NO: 24as disclosed in WO2012079000A1, which sequence is explicitlyincorporated herein for use in the present invention and possibleinclusion in one or more claims herein. In an example, the CD3 zetasignaling domain is encoded by SEQ ID NO: 2, i.e., the nucleic acidsequence of SEQ ID NO: 18 as disclosed in WO2012079000A1, which sequenceis explicitly incorporated herein for use in the present invention andpossible inclusion in one or more claims herein.

For example, the first signalling domain is a human CD28 domain and thecell population of the invention is a population of human cells eachcomprising an endogenous nucleotide sequence encoding said human CD28domain.

For example, the first signalling domain is a human 4-1BB domain and thecell population of the invention is a population of human cells eachcomprising an endogenous nucleotide sequence encoding said human 4-1BBdomain.

For example, the first signalling domain is a human OX40 domain and thecell population of the invention is a population of human cells eachcomprising an endogenous nucleotide sequence encoding said human OX40domain.

In an example, the first signalling domain is a CD3 domain, and thefirst and second intracellular signalling domains do not naturally occurtogether in a single cell (eg, a human wild-type cell or a cell isolatedfrom the patient), eg, the second domain is a CD28, CD27, OX40 or 4-1BBdomain.

In an example, the first intracellular domain is a CD3 domain, CD28domain or 4-1BB domain.

In an example, the CAR is an engineered single polypeptide comprising(in N- to C-terminal direction) an antigen binding site (eg, an antibodyscFv, which may be human); an optional hinge (eg, a human CD8a hinge); atransmembrane domain (eg, a human CD8a or CD28 transmembrane domain);and a human CD3 domain. In an example, the CAR is a complex of two ormore of said polypeptides. Optionally, the CAR comprises a furtherintracellular signalling domain (i) between the transmembrane and CD3domains. Optionally, the CAR comprises a further intracellularsignalling domain, wherein the CD3 domain is between the furthersignaling domain and the transmembrane domain. In an example, thefurther signalling domain is a human CD27 domain, CD28 domain, ICOSdomain, OX40 domain, CD40 domain, 4-1BB domain, a FcεRIγ domain, CD64domain or CD16 domain. In an alternative, instead of a singlepolypeptide, the CAR comprises an engineered complex of at least 2polypeptides comprising said domains.

The immune cells may be administered either alone, or as apharmaceutical composition in combination with diluents and/or withother components such as IL-2 or other cytokines or cell populations.

In an embodiment, the immune cells (eg, CAR cells or cells bearing TCRs)comprise cell surface binding sites (eg, provided by the CAR or TCR)that bind a TAA. Tumour antigens (TAA) are proteins that are produced bytumour cells that elicit an immune response, particularly T-cellmediated immune responses. The selection of the antigen bindingspecificity will depend on the particular type of cancer to be treated.Tumour antigens are well known in the art and include in the context ofan embodiment of the invention, for example, a glioma-associatedantigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin,alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulm, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinalcarboxyi esterase, mut hsp70-2, M-CSF, prostase, prostate-specificantigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2/neu,survivin and telomerase, prostate-carcinoma tumour antigen-1 (PCTA-1),MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor(IGF)-I, IGF-II, IGF-I receptor and mesothelin.

In one embodiment, the tumour antigen comprises one or more antigeniccancer epitopes associated with a malignant tumour. Malignant tumoursexpress a number of proteins that can serve as target antigens for animmune attack. These molecules include but are not limited totissue-specific antigens such as MART-1, tyrosinase and GP 100 inmelanoma and prostatic acid phosphatase (PAP) and prostate-specificantigen (PSA) in prostate cancer. Other target molecules belong to thegroup of transformation-related molecules such as the oncogene HER-2/NeuErbB-2. Yet another group of target antigens are onco-foetal antigenssuch as carcinoembryonic antigen (CEA). In B-cell lymphoma thetumour-specific idiotype immunoglobulin constitutes a trulytumour-specific immunoglobulin antigen that is unique to the individualtumour. B-cell differentiation antigens such as CD I 9, CD20 and CD37are other candidates for target antigens in B-cell lymphoma. Some ofthese antigens (CEA, HER-2, CD19, CD20, idiotype) have been used astargets for passive immunotherapy with monoclonal antibodies withlimited success. The first antigen or fourth binding moiety can be anyof these TAAs or can be an antigenic sequence of any of these TAAs.

Non-limiting examples of TAA antigens in an embodiment of the inventioninclude the following: Differentiation antigens such as MART-1/MelanA(MART-1), g 1 OO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumour-specificmultilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pi5; overexpressed embryonic antigens such as CEA; overexpressed oncogenesand mutated tumour-suppressor genes such as p53, Ras, HER-2/neu; uniquetumour antigens resulting from chromosomal translocations; such asBCR-ABL, E2A-PRL, H4-RET, 1GH-IGK, MYL-RAR; and viral antigens, such asthe Epstein Barr virus antigens EBVA and the human papillomavirus (HPV)antigens E6 and E7. Other large, protein-based antigens include TSP-180,MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl 85erbB2, p 1 80erbB-3, c-met,nm-23H 1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras,beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4(791Tgp72}alpha-fetoprotem, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA,CA 195, CA 242, CA-50, CAM43, CD68\ I, CO-029, FGF-5, G250, Ga733VEpCAM,HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS 1,SDCCAG16, TA-90\Mac-2 binding proteiiAcyclophilin C-associated protein,TAAL6, TAG72, TLP, and TPS.

In one embodiment, the CAR or TCR comprises a binding site for human CD19, eg, for a CAR this can be provided by an anti-CD 19 scFV, optionallywherein the anti-CD19 scFV is encoded by SEQ ID NO: 3, i.e., SEQ ID: 14disclosed in WO2012079000A1. In one embodiment, the anti-CD 19 scFVcomprises SEQ ID NO: 4, i.e., the amino acid sequence of SEQ ID NO: 20disclosed in WO2012079000A1. The sequences in this paragraph appear inWO2012079000A1 and are explicitly incorporated herein for use in thepresent invention and for possible inclusion in one or more claimsherein.

In one embodiment, the transmembrane domain that naturally is associatedwith one of the domains in the CAR is used. In some instances, thetransmembrane domain can be selected or modified by amino acidsubstitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or from asynthetic source.

Where the source is natural, the domain may be derived from anymembrane-bound or transmembrane protein. Transmembrane regions ofparticular use in this invention may be derived from (i.e. comprise atleast the transmembrane region(s) of) the alpha, beta or zeta chain ofthe T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CDS, CD9, CD16,CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137 or CD 154.Alternatively the transmembrane domain may be synthetic, in which caseit will comprise predominantly hydrophobic residues such as leucine andvaline. Optionally, a triplet of phenylalanine, tryptophan and valinewill be found at each end of a synthetic transmembrane domain.

Optionally, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length forms a linkage between the transmembranedomain and the intracellular part of the immune cell transmembraneprotein, such as the CAR. A glycine-serine doublet provides aparticularly suitable linker (eg, a (G₄S)_(n) linker as disclosedherein).

Optionally, the transmembrane domain is the CD8 transmembrane domainencoded by SEQ ID NO: 5, i.e., the nucleic acid sequence of SEQ ID NO:16 disclosed in WO2012079000A1. In one embodiment, the CD8 transmembranedomain comprises SEQ ID NO: 6, i.e., the amino acid sequence of SEQ IDNO: 22 disclosed in WO2012079000A1. The sequences in this paragraphappear in WO2012079000A1 and are explicitly incorporated herein for usein the present invention and for possible inclusion in one or moreclaims herein.

In some instances, the transmembrane domain comprises the CD8 hingedomain encoded by SEQ ID NO: 7, i.e., the nucleic acid sequence of SEQID NO: 15 disclosed in WO2012079000A1. In one embodiment, the CD8 hingedomain comprises SEQ ID NO: 8, i.e., the amino acid sequence of SEQ IDNO: 21 disclosed in WO2012079000A1. The sequences in this paragraphappear in WO2012079000A1 and are explicitly incorporated herein for usein the present invention and for possible inclusion in one or moreclaims herein.

The intracellular part or otherwise the intracellular signalingdomain(s) of the transmembrane protein expressed by cells of the cellpopulation administered to the patient is responsible for activation ofat least one of the normal effector functions of the immune cell thatexpresses the transmembrane protein (eg, a T-cell function, such asleading to cytotoxicity (for T-effector cells for example) orsuppression (for T-regulatory cells)). The term “effector function”refers to a specialized function of a cell. Effector function of a Tcell, for example, may be cytolytic activity or helper activityincluding the secretion of cytokines. Thus the term “intracellularsignaling domain” refers to the portion of a protein which transducesthe effector function signal and directs the cell to perform aspecialized function. While usually the entire intracellular signalingdomain can be employed, in many cases it is not necessary to use theentire chain. To the extent that a truncated portion of theintracellular signaling domain is used, such truncated portion may beused in place of the intact chain as long as it transduces the effectorfunction signal. The term “signaling domain” is thus meant to includeany truncated portion of the intracellular signaling domain sufficientto transduce the effector function signal. Examples of intracellularsignaling domains for use in the transmembrane protein of theadministered cells include the cytoplasmic sequences of the T cellreceptor (TCR) and co-receptors that act in concert to initiate signaltransduction following antigen receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability.

It is known that signals generated through the TCR alone areinsufficient for full activation of the T cell and that a secondary orco-stimulatory signal is also required. Thus, T cell activation can besaid to be mediated by two distinct classes of cytoplasmic signalingsequence: those that initiate antigen-dependent primary activationthrough the TCR (primary cytoplasmic signaling domain) and those thatact in an antigen-independent manner to provide a secondary orco-stimulatory signal (secondary cytoplasmic signaling domain) Primarycytoplasmic signaling sequences regulate primary activation of the TCRcomplex either in a stimulatory way, or in an inhibitory way. Primarycytoplasmic signaling sequences that act in a stimulatory manner maycontain signaling motifs which are known as immunoreceptortyrosine-based activation motifs or ITAMs.

In an example, the first signalling domain is a primary cytoplasmicsignaling domain (eg, CD3 domain) In an example, the first signallingdomain is a secondary cytoplasmic signaling domain (eg, CD28 or 4-1BBdomain).

In an example, the first signalling domain comprises one or more ITAMs.

Examples of suitable ITAM containing primary cytoplasmic signalingdomains that are of particular use in the invention include thosederived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3epsilon, CDS, CD22, CD79a, CD79b, and CD66d. It is particularlypreferred that cytoplasmic signaling molecule in the transmembraneprotein of the invention comprises a cytoplasmic signaling sequencederived from CD3 zeta.

The intracellular part optionally comprises (eg, as the first signallingdomain or a further intracellular domain) a domain of a costimulatorymolecule. A costimulatory molecule is a cell surface molecule other thanan antigen receptor or their ligands that is required for an efficientresponse of lymphocytes (eg, T- or NK cells) to an antigen. Examples ofsuch molecules include CD27, CD28, 4-1BB (CD 137), OX40, CD30, CD40,PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7,LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, andthe like. Thus, these and other costimulatory elements are within thescope of the invention for use in the intracellular part of thetransmembrane protein.

The intracellular moiety domains may be linked together by one or morelinkers, eg, a (G₄S)_(n) linker as disclosed herein.

In one embodiment, the intracellular moiety comprises the signalingdomain of CD3-zeta and the signaling domain of CD28. In anotherembodiment, the intracellular moiety comprises the signaling domain ofCD3-zeta and the signaling domain of 4-1BB. In yet another embodiment,the intracellular moiety comprises the signaling domain of CD3-zeta andthe signaling domain of CD28 and 4-1BB.

In one embodiment, the intracellular moiety comprises the signalingdomain of 4-1BB and the signaling domain of CD3-zeta, wherein thesignaling domain of 4-1BB is encoded by SEQ ID NO: 9, i.e., the nucleicacid sequence set forth in SEQ ID NO: 17 disclosed in WO2012079000A1 andthe signaling domain of CD3-zeta is encoded by SEQ ID NO: 2, i.e., thenucleic acid sequence set forth in SEQ ID NO: 18 disclosed inWO2012079000A1. The sequences in this paragraph appear in WO2012079000A1and are explicitly incorporated herein for use in the present inventionand for possible inclusion in one or more claims herein.

In one embodiment, the intracellular moiety comprises the signalingdomain of 4-1BB and the signaling domain of CD3-zeta, wherein thesignaling domain of 4-1BB comprises SEQ ID NO: 10, i.e., the amino acidsequence of SEQ ID NO: 23 disclosed in WO2012079000A1 and the signalingdomain of CD3-zeta comprises SEQ ID NO: 1, i.e., the amino acid sequenceof SEQ ID NO: 24 disclosed in WO2012079000A1. The sequences in thisparagraph appear in WO2012079000A1 and are explicitly incorporatedherein for use in the present invention and for possible inclusion inone or more claims herein.

In one embodiment, the intracellular moiety comprises the signalingdomain of 4-1BB and the signaling domain of CD3-zeta, wherein thesignaling domain of 4-1BB comprises SEQ ID NO: 10, i.e., the amino acidsequence set forth in SEQ ID NO: 23 as disclosed in WO2012079000A1 andthe signaling domain of CD3-zeta comprises SEQ ID NO: 1, i.e., the aminoacid sequence set forth in SEQ ID NO: 24 disclosed in WO2012079000A1.The sequences in this paragraph appear in WO2012079000A1 and areexplicitly incorporated herein for use in the present invention and forpossible inclusion in one or more claims herein.

Sources of T-cells and other immune cells are disclosed inWO2012079000A1, U.S. Pat. Nos. 8,906,682, 8,911,993, 8,916,381,8,975,071, 9,101,584, 9,102,760, 9,102,761, 9,328,156, 9,464,140,9,481,728, 9,499,629, 9,518,123, 9,540,445, 2013/0287748, 2013/0288368,2013/0309258, 2014/0106449, 2014/0370017, 2015/0050729, 2015/0093822,2015/0099299, 2015/0118202, 2016/0130355, 2016/0159907, 2016/0194404,2016/0208012, as well as methods of generating, activating and expandingthese. These disclosures are referred to for possible use in working thepresent invention.

Cancers for Treatment or Prevention by the Method

Cancers that may be treated include tumours that are not vascularized,or not substantially vascularized, as well as vascularized tumours. Thecancers may comprise non-solid tumours (such as haematological tumours,for example, leukaemias and lymphomas) or may comprise solid tumours.Types of cancers to be treated with the invention include, but are notlimited to, carcinoma, blastoma, and sarcoma, and certain leukaemia orlymphoid malignancies, benign and malignant tumours, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers andpaediatric tumours/cancers are also included.

Haematologic cancers are cancers of the blood or bone marrow. Examplesof haematological (or haematogenous) cancers include leukaemias,including acute leukaemias (such as acute lymphocytic leukaemia, acutemyelocytic leukaemia, acute myelogenous leukaemia and myeloblasts,promyeiocytic, myelomonocytic, monocytic and erythroleukaemia), chronicleukaemias (such as chronic myelocytic (granulocytic) leukaemia, chronicmyelogenous leukaemia, and chronic lymphocytic leukaemia), polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent andhigh grade forms), multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, myeiodysplastic syndrome, hairy cell leukaemia andmyelodysplasia.

Solid tumours are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumours can be benign or malignant.Different types of solid tumours are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumours, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer,testicular tumour, seminoma, bladder carcinoma, melanoma, and CNStumours (such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medu!loblastoma, Schwannoma craniopharyogioma,ependymoma, pineaioma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

In one embodiment, the administered cells express a first antigenbinding site (eg, comprised by a CAR) that is designed to treat aparticular cancer. For example, it specifically binds to CD19 can beused to treat cancers and disorders, eg, pre-B ALL (paediatricindication), adult ALL, mantle cell lymphoma, diffuse large B-celllymphoma or for salvage post allogenic bone marrow transplantation. Inanother embodiment, the first moiety or first binding site specificallybinds CD22 to treat diffuse large B-cell lymphoma.

In one embodiment, cancers and disorders include but are not limited topre-B ALL (paediatric indication), adult ALL, mantle cell lymphoma,diffuse large B-cell lymphoma, salvage post allogenic bone marrowtransplantation, and the like can be treated using a combination ofbridging agents (or binding moieties or sites comprised by a singleagent) that target two or three of: CD19, CD20, CD22, and ROR1 (eg, CD19and one of the other targets).

In an example, the cells comprises first and second transmembraneproteins (eg, CARs or a CAR and an engineered TCR expressed by a T-cell)that are different, eg that differ in their target antigens (andoptionally otherwise are the same). Similarly, the invention may use amixture of immune cells (eg, a mixture of CAR-cells), eg comprised bythe same transplant, wherein the mixture comprises cells comprisingtransmembrane proteins (eg, CARs or a CAR and an engineered TCRexpressed by a T-cell) that are different, eg that differ in theirtarget antigens (and optionally otherwise are the same). This may beuseful for reducing resistance to treatment by cancers, for example, ormore effectively targeting cell populations such as cancer cells thatsurface express a plurality of target antigens.

In one embodiment, the antigen binding site specifically binds tomesothelin to treat or prevent mesothelioma, pancreatic cancer orovarian cancer.

In one embodiment, the antigen binding site specifically binds toCD33/IL3Ra to treat or prevent acute myelogenous leukaemia.

In one embodiment, the antigen binding site specifically binds to c-Metto treat or prevent triple negative breast cancer or non-small cell lungcancer.

In one embodiment, the antigen binding site specifically binds to PSMAto treat or prevent prostate cancer.

In one embodiment, the antigen binding site specifically binds toGlycolipid F77 to treat or prevent prostate cancer.

In one embodiment, the antigen binding site specifically binds toEGFRvIlI to treat or prevent gliobastoma.

In one embodiment, the antigen binding site specifically binds to GD-2to treat or prevent neuroblastoma or melanoma.

In one embodiment, the antigen binding site specifically binds toNY-ESO-1 TCR to treat myeloma, sarcoma or melanoma.

In one embodiment, the antigen binding site specifically binds to MAGEA3 TCR to treat myeloma, sarcoma and melanoma.

Specific antigen binding is binding with a KD of 1 mM or lower (eg, 1 mMor lower, 100 nM or lower, 10 nM or lower, 1 nM or lower, 100 pM orlower, or 10 pM or lower) as determined by Surface Plasmon Resonance(SPR) in vitro at 25 degrees celcius or rtp.

In one example, said treatment using the method reduces progression ofthe disease or condition or a symptom thereof. In one example, saidtreatment using the method reduces incidence of the disease or conditionor symptom thereof, eg, for at least 1, 2, 3, 4, or 5 years.

In an example, the method is in vivo in a mammal, eg, a human, man orwoman, or male child or female child, or a human infant (eg, no morethan 1, 2, 3 or 4 years of age). In an example, the patient is an adulthuman or a paediatric human patient.

The CAR or TCR is engineered, ie, comprises a non-naturally-occurringcombination of moieties and domains. In an example, the cell therapytargets a target cell, wherein the target cell is a cancer cell, eg, aleukaemic cell, lymphoma cell, adenocarcinoma cell or cancer stem cell.Optionally, the CAR or TCR of administered immune cells specificallybinds to human CD19 (and optionally the target cell is a leukaemic orlymphoma cell), EpCAM (and optionally the target cell is a lung cancercell, gastrointestinal cancer cell, an adenocarcinoma, cancer stemcell), CD20 (and optionally the target cell is a leukaemic cell), MCSP(and optionally the target cell is a melanoma cell), CEA, EGFR,EGFRvIII, sialyl Tn, CD133, CD33 (and optionally the target cell is aleukaemic cell, eg, AML cell), PMSA, WT1, CD22, L1CAM, ROR-1, MUC-16,CD30, CD47, CD52, gpA33, TAG-72, mucin, CIX, GD2, GD3, GM2, CD123,VEGFR, integrin, cMET, Her1, Her2, Her3, MAGE1, MAGE A3 TCR, NY-ESO-1,IGF1R, EPHA3, CD66e, EphA2, TRAILR1, TRAILR2, RANKL, FAP, Angiopoietin,mesothelin, Glycolipid F77 or tenascin.

Optionally, the CAR comprises the variable domains of an antibodyselected from the group consisting of the CD19 binding site ofblinatumomab or antibody HD37; EpCAM binding site of Catumaxomab; CD19binding site of AFM11; CD20 binding site of Lymphomun; Her2 binding siteof Ertumaxomab; CEA binding site of AMG211 (MEDI-565, MT111); PSMAbinding site of Pasotuxizumab; EpCAM binding site of solitomab; VEGF orangiopoietin 2 binding site of RG7221 or RG7716; Her1 or Her3 bindingsite of RG7597; Her2 or Her3 binding site of MM111; IGF1R or Her3binding site of MM141; CD123 binding site of MGD006; gpa33 binding siteof MGD007; CEA binding site of TF2; CD30 binding site of AFM13; CD19binding site of AFM11; and Her1 or cMet binding site of LY3164530.

Optionally, the CAR comprises the variable domains of an antigen bindingsite of an antibody selected from the group consisting of REOPRO®(Abciximab); RITUXAN® (Rituximab); ZENAPAX® (Daclizumab); SIMULECT®(Basiliximab); SYNAGIS® (Palivizumab); REMICADE® (Infliximab);HERCEPTIN® (Trastuzumab); MYLOTARG® (Gemtuzumab ozogamicin); CAMPATH®(Alemtuzumab); ZEVALIN® (Ibritumomab); HUMIRA® (Adalimumab); XOLAIR®(Omalizumab); BEXXAR® (Tositumomab); RAPTIVA® (Efalizumab); ERBITUX®(Cetuximab); AVASTIN® (Bevacizumab); TYSABRI® (Natalizumab); ACTEMRA®(Tocilizumab); VECTIBIX® (Panitumumab); LUCENTIS® (Ranibizumab);SOLIRIS® (Eculizumab); CIMZIA® (Certolizumab); SIMPONI® (Golimumab);ILARIS® (Canakinumab); STELARA® (Ustekinumab); ARZERRA® (Ofatumumab);PROLIA® (Denosumab); NUMAX® (Motavizumab); ABTHRAX® (Raxibacumab);BENLYSTA® (Belimumab); YERVOY® (Ipilimumab); ADCETRIS® (Brentuximabvedotin); PERJETA® (Pertuzumab); KADCYLA® (Ado-trastuzumab); and GAZYVA®(Obinutuzumab).

In an example, the target cell is a blood cell, eg, a stem cell or bonemarrow cell of a human or animal. Optionally, the target cell is a B- orT-cell.

In an example, the CAR or TCR comprises an antigen binding site for anautoimmune disease target and the signaling down-regulates cytotoxicactivity or proliferation of the immune cells. The term “autoimmunedisease” as used herein is defined as a disorder that results from anautoimmune response. An autoimmune disease is the result of aninappropriate and excessive response to a self-antigen. Examples ofautoimmune diseases include but are not limited to, Addision's disease,alopecia greata, ankylosing spondylitis, autoimmune hepatitis,autoimmune parotitis, Crohn's disease, diabetes (Type I), dystrophicepidermolysis bullosa, epididymitis, glomerulonephritis, Graves'disease, Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia,systemic lupus erythaematosus, multiple sclerosis, myasthenia gravis,pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis,sarcoidosis, scleroderma, Sjogren's syndrome, spondyloarthropathies,thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia,ulcerative colitis, among others.

Within the overall memory T cell population, several distinctsubpopulations have been described and can be recognised by thedifferential expression of chemokine receptor CCR7 and L-selectin(CD62L). Stem memory T_(SCM) cells, like naive cells, are CD45RO−,CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+ and IL-7Rα+, but theyalso express large amounts of CD95, IL-2Rβ, CXCR3, and LFA-1, and shownumerous functional attributes distinctive of memory cells. Centralmemory T_(CM) cells express L-selectin and the CCR7, they secrete IL-2,but not IFNγ or IL-4. Effector memory T_(EM) cells, however, do notexpress L-selectin or CCR7 but produce effector cytokines like IFNγ andIL-4. Memory T-cells, such as T_(SCM) may be particularly useful forestablishing a sustained population of engineered immune cells in thehuman.

Any immune cell, target cell or stem cell herein can, in an example, bea T_(SCM), T_(CM) or T_(EM) cell, eg, a human T_(SCM), T_(CM) or T_(EM)cell. In an example, the immune cells of the cell therapy (eg, CAR-Tcells) each is a progeny of a cell of a human suffering from anautoimmune disease, an inflammatory disease, a viral infection or acancer, eg, wherein the human is suffering from lymphoblastic leukaemia,ALL (eg, T-ALL), CLL (eg, B-cell chronic lymphocytic leukaemia) ornon-Hodgkin's lymphoma. The human may, for example, be the patient or arelative (eg, sibling or parent) thereof.

In an example, the administered immune cells have been engineered forenhanced signaling, wherein the signaling is selected from CD28, 4-1BB,OX40, ICOS and CD40 signaling.

Optionally, the target cells (eg, tumour cells) are killed. In anexample, each target cell is a tumour cell and the method treats orreduces the risk of cancer, or treats or reduces the risk of cancerprogression in the human.

Optionally, the human has cancer. In an example, the cancer is ahaematological cancer. In an example, the human has a cancer of B-cellorigin. In an example, the human has a cancer of T-cell origin. Forexample the cancer is lung cancer, melanoma, breast cancer, prostatecancer, colon cancer, renal cell carcinoma, ovarian cancer,neuroblastoma, rhabdomyosarcoma, leukaemia and lymphoma. Preferredcancer targets for use with the present invention are cancers of B cellorigin, particularly including acute lymphoblastic leukaemia, B-cellchronic lymphocytic leukaemia or B-cell non-Hodgkin's lymphoma. In anexample, the cancer is a cancer of T-cell or B-cell origin, eg,lymphoblastic leukaemia, ALL (eg, T-ALL), CLL (eg, B-cell chroniclymphocytic leukaemia) or non-Hodgkin's lymphoma.

Optionally, each administered immune cell (eg, CAR-cells) is a progenyof an immune cell of said human, eg, wherein the human is suffering fromlymphoblastic leukaemia, Diffuse Large B-cell Lymphoma (DLBCL), ALL (eg,T-ALL or B-ALL), CLL (eg, B-cell chronic lymphocytic leukaemia) ornon-Hodgkin's lymphoma. Optionally, each administered immune cell (eg,CAR-cells) is an autologous cell (eg, T-cell) of said human or is aprogeny of such an autologous cell. As used herein, the term“autologous” is meant to refer to any material derived from the sameindividual to which it is later to be re-introduced into the individual.“Allogeneic” refers to a graft derived from a different animal of thesame species.

Optionally, each administered immune cell (eg, CAR-cells) is derivedfrom a blood or tumour sample of the human and activated and expanded invitro before step (c). “Activation,” as used herein, refers to the stateof a T-cell or other immune cell that has been sufficiently stimulatedto induce detectable cellular proliferation. Activation can also beassociated with induced cytokine production, and detectable effectorfunctions. The term “activated T cells” refers to, among other things, Tcells that are undergoing cell division.

In an embodiment, the human has an autoimmune disease, wherein theimmune cells that are administered (eg, CAR-cells) are anergic, or havereduced proliferation and/or cytotoxic activity when bound to targetcells, whereby the cell transplant cells (and/or their progeny) competewith endogenous immune cells of said human that up-regulate saidautoimmune disease.

The administration of immune cells in the method may be by cell infusioninto the blood of the patient. The immune cells may be expanded toproduce an expanded immune cell population that is administered to thepatient. The immune cells may be activated produce an activated immunecell population that is administered to the patient. In methods herein,an effective amount of immune cells are administered. An “effectiveamount” as used herein, means an amount which provides a therapeutic orprophylactic benefit to treat or prevent the disease or condition.

In an embodiment of the method of the invention, the method treats orreduces the risk of cancer in a patient (eg, a human), wherein thepatient has undergone lymphodepletion before administration of theimmune cells to the patient.

In one embodiment, the human is resistant to at least onechemotherapeutic agent.

In one embodiment, the chronic lymphocytic leukaemia is refractory CD19+ leukaemia and lymphoma.

The invention also includes a method of generating a persistingpopulation of genetically engineered T cells in a human diagnosed withcancer, wherein the administered cells comprise T-cells and thepersisting population comprises progeny thereof. In one embodiment, themethod comprises administering to a human a T-cell population (eg, a CART-cell population), wherein the persisting population of geneticallyengineered T-cells persists in the human for at least one month afteradministration. In one embodiment, the persisting population ofgenetically engineered T-cells comprises a memory T-cell. In oneembodiment, the persisting population of genetically engineered T-cellspersists in the human for at least three months after administration. Inanother embodiment, the persisting population of genetically engineeredT-cells persists in the human for at least four months, five months, sixmonths, seven months, eight months, nine months, ten months, elevenmonths, twelve months, two years, or three years after administration.

In one embodiment, the chronic lymphocytic leukaemia is treated. Theinvention also provides a method of expanding a population of theengineered T-cells or NK cells in a human diagnosed with cancer, whereinthe administered cells comprise T-cells and/or NK cells and the expandedpopulation comprises progeny thereof.

Optionally, autologous lymphocyte infusion is used in the treatment. Forexample, autologous PBMCs are collected from a patient in need oftreatment and CAR-T-cells are engineered to express the CARtransmembrane protein, activated and expanded using the methods known inthe art and then infused back into the patient in step (a).

In an example, the administered cells are pluripotent or multipotent.

The stem cell cannot develop into a human. In an embodiment, the stemcell cannot develop into a human embryo or zygote.

In an example, the administered cell population comprises bone marrowstem cells, eg, human autologous or allogeneic cells.

In an example, the administered cell population comprises haematopoieticstem cells, eg, human autologous or allogeneic cells.

Modifying Microbiota

Medical practice often involves the administration of antibiotics topatients. Such treatments can typically involve administration ofbroad-spectrum antibiotics, or antibiotics that target manygram-positive bacterial species or many gram-negative species withoutdiscrimination. Similarly, use of broad-spectrum antibiotics in farmingand agriculture, for example, raise environmental concerns, includingentry of such antibiotics into the human and animal food chain which maybe deleterious to health and may add to development of microbialresistance. Rather, in an example, the invention involves selectivetargeting of a first microbial (eg, bacterial or archaeal) species orstrain of the microbiota. As shown in the worked examples herein,selective targeting of a particular bacterial species has been achievedusing guided nuclease targeting of the genome of the selected species,whilst at the same time sparing related species and strains, as well asspecies that co-reside (in the Examples species that co-reside in humangut microbiota). Thus, in one example, the step of causing dysbiosis orstep (b) comprises killing first cells of a microbiota sub-population orinhibiting growth of said sub-population by using guided nuclease (eg,RNA guided nuclease) targeting to the genome of first cells comprised bya microbiota sub-population. Suitable systems for carrying out theguided nuclease targeting are, for example, engineered CRISPR/Cassystems, TALENs, meganucleases and zinc finger systems. By way ofexample, CRISPR/Cas-mediated guided targeting of a selected human gutmicrobiota bacterial species in a consortium is demonstrated in theExamples herein. The targeting produces nuclease cutting of targetspecies or strain DNA, for example, which reduces the relativeproportion of the species in the mircrobiota or inhibits growth of thesub-population of said species in the microbiota. Selective targeting ofspecies in the method is generally advantageous to enable finer controlover change in the relative proportions of bacterial and/or archaealspecies in the microbiota. In this way, the invention provides theability to alter the microbiota with the aim of influencing theupregulation or downregulation of particular immune cell populations,such as T_(H)1, T_(H)17 and/or T_(reg) cells (be these cells endogenousto the patient and/or comprised by adoptive immune cell populations thatare administered to the patient), or other outcomes of modulating themicrobiota as described herein.

In an example first cell population growth is reduced by at least 5-foldcompared to the growth before said dysbiosis or step (b). The method maycomprise inhibiting first cell population growth on a gut surface. Themethod may comprise inhibiting first cell population growth on a plant(eg, leaf and/or stem) surface.

In an alternative, instead of being applied to a subject, the treatmentis applied to an environment or soil (eg, the treatment is a fertiliser,plant growth promoting or inhibiting, herbicide or pesticide treatment),wherein the treatment is modulated by the invention.

It will be readily apparent to the skilled addressee how to determinechanges in bacteria and archaea in a gut microbiota or other microbiota.For example, this can be done by analyzing a facecal sample of thepatient before and after the treatment. One may determine the types ofdifferent species or strains in each sample and the proportion ofspecies or strains before and after treatment. Using conventionalanalysis of 16s ribosomal RNA-encoding DNA (16s rDNA) it is possible toidentify species, for example. Additionally or alternatively, standardbiochemical test can be used to identify strains or species, eg, alsoinvolving one or more of: staining, motility testing, serologicaltesting, phage typing and identification disc testing (eg using a KirbyBaur disc diffusion method). Biochemical testing may involve one or moreof: a (a) Catalase test (b) Coagulase test (c) Oxidase test (d) Sugarfermentation test (e) Indole test (f) Citrate test and (g) Urease test.Relative proportions may be determined by growing colonies on agarplates (as in the Examples herein) from each sample and counting colonynumbers.

In an example, the dysbiosis or step (b) increases the proportion ofBacteroides (eg, B fragalis and/or B thetaiotamicron) in the microbiota,eg, gut microbiota.

In an example, the dysbiosis or step (b) decreases the proportion ofBacteroides (eg, B fragalis and/or B thetaiotamicron) in the microbiota,eg, gut microbiota. In an example, the dysbiosis or step (b) increasesthe proportion of Bacteroidetes to Firmicutes in the microbiota, eg, gutmicrobiota. In an example, the dysbiosis or step (b) decreases theproportion of Bacteroidetes to Firmicutes in the microbiota, eg, gutmicrobiota.

Accumulating evidence supports the role of commensal strains ofBifidobacterium and Clostridium spp. belonging to clusters IV and XIVain the induction of Treg cells. See, eg, Lopez et al. In an example, thedysbiosis or step (b) reduces the proportion of one or more Clostridiumspecies or strain (eg, In an example, each species is a cluster IV orXIVa Clostridium species) in the gut microbiota. In an example, thedysbiosis or step (b) increases the proportion of one or moreClostridium species or strain (eg, In an example, each species is acluster IV or XIVa Clostridium species) in the gut microbiota.

In an example, the dysbiosis or step (b) reduces the proportion ofBifidobacterium (eg, B bifidum) in the gut microbiota. In an example,the dysbiosis or step (b) increases the proportion of Bifidobacterium(eg, B bifidum) in the gut microbiota.

For example, by selectively altering the human gut microbiota theinvention provides for upregulation of CAR-T or other ACT treatment (eg,wherein the altered microbiota downregulates T_(reg) cells in thepatient that has received the CAR-T or ACT administration and/orupregulates T_(H)1 and/or T_(H)17 cells in the patient—such cells beingcomprised by the CAR-T or ACT transplant for example). DownregulatingT_(reg) cells may reduce suppression of T-effectors and/or T-helpers inthe patient, thereby enhancing the CAR-T or ACT cytotoxicity or otherdesirable activity against cancer or other disease-mediating cells.Upregulating T_(H)1 and/or T_(H)17 cells may increase T-effectoractivity, thereby enhancing the CAR-T or ACT cytotoxicity or otherdesirable activity against cancer or other disease-mediating cells.

In another example, alteration of the microbiota can be used as a switchto dampen down CAR-T or other ACT treatment (eg, wherein the alteredmicrobiota upregulates T_(reg) cells in the patient that has receivedthe CAR-T or ACT administration and/or downregulates T_(H)1 and/orT_(H)17 cells in the patient—such cells being comprised by the CAR-T orACT transplant for example). Upregulating T_(reg) cells may increasesuppression of T-effectors and/or T-helpers in the patient, therebyreducing the CAR-T or ACT ability to promote cytokine release or otherundesirable activity. Downregulating T_(H)1 and/or T_(H)17 cells maydecrease T-effector activity, thereby reducing the CAR-T or ACT abilityto promote cytokine release or other undesirable activity. This may beuseful for limiting the risk of cytokine release syndrome (CRS) in thepatient. Subsequent further modification of the gut microbiota of thepatient using the method of the invention can be performed to upregulatethe CAR-T or ACT treatment when it is desired to use this once more toaddress the disease or condition at hand (eg, a cancer, such as ahaematological cancer). In this instance, memory T-cell CAR-T or ACTpopulations may be present in the patient from the earlier treatment,and the upregulation using microbiota alteration according to theinvention may upregulate memory T-cells to differentiate into effectorand/or helper cells to address the disease or condition. Thus, in oneexample, the cell therapy of the invention comprises administering animmune cell population comprising immune memory cells (eg, memoryT-cells, such as central memory T cells (T_(CM)) and/or stem cell memorycells (T_(SCM)); and/or the administered population comprises cells thatspawn such memory cells following the initial microbiota alteration.

Whilst one aspect of the invention recognizes utility for modulatingcell-based therapy in a patient, another aspect recognizes utility formodulating (ie, treating or preventing) cell-mediated diseases andconditions in patients, such as autoimmune and inflammatory diseases andconditions which are mediated, for example by T-cells or other immunecells of the patient. In a further aspect, the invention recognizesutility as a means for modulating (eg, enhancing) another therapy of adisease or condition; for example, for enhancing or effecting therapywith an antibody or anti-viral medicine to treat or prevent the diseaseor condition. For example, the medicine can be an immune checkpointantagonist or agonist (eg, for treating or preventing a cancer, such asmelanoma or NSCLC). By “effecting therapy” it is contemplated that thepatient does not respond or poorly responds to the medicine and themicrobiota alteration according to the invention (eg, using selectiveguided nuclease targeting of a bacterial or archaeal species asdescribed herein) brings about a response (or improved response) to themedicine by the patient. For example, the method of the inventionupregulates T_(H)17 cells in a patient suffering from HIV infection. Inone aspect, this enhances anti-retroviral therapy or HIV vaccine therapyof the patient. The T_(H)17 cells may be the patient's endogenous cellsor cells provided by ACT of the patient. In another example, the methodof the invention upregulates T_(H)17 cells in a patient suffering from acancer (eg, melanoma or lung cancer, such as NSCLC). In one aspect, thisenhances immune checkpoint antagonism or agonism therapy of the patient.The T_(H)17 cells may be the patient's endogenous cells or cellsprovided by ACT of the patient. For example, the therapy is antibodytherapy using an antibody selected from ipilimumab (or YERVOY™),tremelimumab, nivolumab (or OPDIVO™), pembrolizumab (or KEYTRUDA™),pidilizumab, BMS-936559, durvalumab and atezolizumab.

The invention relates to guided nuclease systems (eg, engineeredCRISPR/Cas systems, TALENs, meganucleases and zinc finger systems),arrays (eg, CRISPR arrays), cRNAs, gRNAs and vectors (eg, phagecomprising components of a said system) for use in a method of theinvention for targeting the first cells or causing said dysbiosis byinhibiting bacterial or archaeal cell population growth or altering therelative proportion of one or more sub-populations of cells in plant,yeast, environmental, soil, human or animal microbiota, such as for thealteration of the proportion of Bacteroidetes (eg, Bacteroides),Firmicutes and/or gram positive or negative bacteria in gut microbiotaof a human. The invention, for example, involves modifying (eg, cuttingand/or mutating) one or more target genomic or episomal nucleotidesequences of a host bacterial cell, eg, a Bacteroidetes cell orFirmicutes cell, or a host archaeal cell. In an example, the firstbacteria are pathogenic gut bacteria.

There have been a number of studies pointing out that the respectivelevels of the two main intestinal phyla, the Bacteroidetes and theFirmicutes, are linked to obesity, both in humans and in germ-free mice.The authors of the studies deduce that carbohydrate metabolism is theimportant factor. They observe that the microbiota of obese individualsare more heavily enriched with bacteria of the phylum Firmicutes andless with Bacteroidetes, and they surmise that this bacterial mix may bemore efficient at extracting energy from a given diet than themicrobiota of lean individuals (which have the opposite proportions). Insome studies, they found that the relative abundance of Bacteroidetesincreases as obese individuals lose weight and, further, that when themicrobiota of obese mice are transferred to germfree mice, these micegain more fat than a control group that received microbiota from leanmice. See, eg, Turnbaugh, P. J., R. E. Ley, M. A. Mahowald, V. Magrini,E. R. Mardis, and J. I. Gordon. 2006, “An obesity-associated gutmicrobiome with increased capacity for energy harvest”, Nature444:1027-1131. In a further aspect, the invention recognizes utility asa means for enhancing an anti-obesity therapy of a patient, eg, byincreasing the ratio of Bacteroidetes versus Firmicutes in themicrobiota.

Optionally the first cells are in the presence of cells of a differentstrain or species, wherein the different cells are Enterobacteriaceae orbacteria that are probiotic, commensal or symbiotic with humans (eg, inthe human gut). In an example, each first cell is a Firmicutes, eg,Streptococcus, cell.

In an example, the invention is able to selectively kill or downregulatethe target microbes in the microbiota whilst not targeting a secondrelated strain of the same species or a different species that isnevertheless phylogenetically related (as indicated by 16s rDNA). Forexample, the microbiota comprises cells of a second bacterial species orstrain, or archaeal species or strain, wherein the second species orstrain has a 16s ribosomal RNA-encoding DNA sequence that is at least80, 85, 90, 95, 96, 97, 98 or 99% identical to an 16s ribosomalRNA-encoding DNA sequence of the first cell species or strain, whereinthe growth of the second cells in the microbiota is not inhibited bysaid method. In an embodiment, the growth of second strain or species isnot inhibited; or the growth of said first cells is inhibited by atleast 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×, 50×, 100× or 1000× the growthinhibition of the second cells.

In one aspect of the method, causing the dysbiosis or step (b) comprisesaltering the proportion of a sub-population of first cells (host cells)in the microbiota, eg, gut microbiota, of the patient, thereby producingan altered gut microbiota that modulates the immune cell therapy in thepatient, wherein the sub-population comprises host cells of said firstspecies or strain, the method comprising using guided nuclease (egRNA-guided nuclease) cutting of a respective target sequence in hostcells to modify the target sequences, whereby host cells are killed orthe host cell population growth is reduced, thereby reducing theproportion of said sub-population in the microbiota. Suitable systemsfor carrying out the guided nuclease cutting are, for example,engineered CRISPR/Cas systems, TALENs, meganucleases and zinc fingersystems. By way of example, CRISPR/Cas-mediated guided cutting of aselected human gut microbiota bacterial species in a consortium isdemonstrated in the Examples herein.

In an example, the target sequence modification is carried out by

-   -   a. combining the microbiota with multiple copies of engineered        nucleic acid sequences encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells,    -   wherein each engineered nucleic acid sequence is operable with a        Cas nuclease in a respective host cell to form a HM-CRISPR/Cas        system and the engineered sequence comprises        -   (i) spacer and repeat sequences encoding a HM-crRNA;        -   (ii) the HM-crRNA comprising a sequence that is capable of            hybridizing to a host cell target sequence to guide Cas            nuclease to the target sequence in the host cell; and    -   optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -   whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said sub-population in the microbiota.

In an alternative, HM-crRNA and tracrRNA are comprised by a single guideRNA (gRNA). In an example, each engineered nucleic acid sequence iscomprised by a respective vector, wherein each vector is optionally aplasmid (eg, a conjugative plasmid capable of transfer into a hostcell), phage, phagemid or prophage. The phage is capable of infecting asaid host cell.

In an example, endogenous Cas nuclease of host cells is used formodification of target nucleotide sequences. In an embodiment,therefore, each vector lacks a Cas (eg, a Cas9) nuclease-encodingsequence. By harnessing endogenous Cas nuclease, embodiments of theinvention use endogenous Cas nuclease activity (ie, without the need forprior genetic modification of the host cell to activate or enhance thenuclease activity). Thus, in an example, the Cas nuclease is encoded bya wild-type gene of the host cell. In an example, the nuclease is activeto achieve the cell killing or growth reduction without inhibition of anendogenous Cas nuclease (or Cas nuclease gene) repressor in the hostcell. Thus, the invention can address wild-type bacterial populationswithout the need for prior manipulation to make bring about effectiveCas-mediated cell killing or growth reduction. Thus, the population canbe exposed to the cRNA when the population is in its wild-typeenvironment (such as comprised by a plant, yeast, environmental, soil,human or animal microbiome).

In an example, the cRNA or gRNA is for administration to (oradministered to) a human or non-human animal patient by mucosal, gut,oral, intranasal, intrarectal or buccal administration.

Optionally said Cas nuclease is provided by an endogenous Type IICRISPR/Cas system of each first cell. Optionally, the tracrRNA sequenceor DNA sequence expressing a tracrRNA sequence is endogenous to eachhost cell. Optionally, each target sequence is comprised by anantibiotic resistance gene, virulence gene or essential gene of therespective host cell, for example the target sequences are identicalbetween the host cells. Optionally, the engineered nucleic acidsequences are comprised by an antibiotic composition, wherein thesequences are in combination with an antibiotic agent (firstantibiotic), and in an example the target sequences are comprised by anantibiotic resistance gene wherein the antibiotic is said firstantibiotic. The antibiotic composition is administered to the patient orsubject to effect said dysbiosis or step (b).

Optionally, each host cell comprises a deoxyribonucleic acid strand witha free end (HM-DNA) encoding a HM-sequence of interest and/or whereinthe method comprising into the host cells such a sequence encoding theHM-DNA, wherein the HM-DNA comprises a sequence or sequences that arehomologous respectively to a sequence or sequences in or flanking thetarget sequence for inserting the HM-DNA into the host genome (eg, intoa chromosomal or episomal site).

The invention also provides vectors for introducing into first cells(host cells) for carrying out the treatment or prevention method of theinvention, wherein each vector is:

An engineered nucleic acid vector for modifying a bacterial or archaealhost cell comprising an endogenous CRISPR/Cas system, the vectorcomprising nucleic acid sequences for expressing a plurality ofdifferent crRNAs (eg, gRNAs) for use in causing the dysbiosis or for usein step (b) of the method; and optionally lacking a nucleic acidsequence encoding a Cas nuclease, wherein a first of said crRNAs iscapable of hybridising to a first nucleic acid sequence in said hostcell; and a second of said crRNAs is capable of hybridising to a secondnucleic acid sequence in said host cell, wherein said second sequence isdifferent from said first sequence; and

-   -   a. the first sequence is comprised by an antibiotic resistance        gene (or RNA thereof) and the second sequence is comprised by an        antibiotic resistance gene (or RNA thereof); optionally wherein        the genes are different;    -   b. the first sequence is comprised by an antibiotic resistance        gene (or RNA thereof) and the second sequence is comprised by an        essential or virulence gene (or RNA thereof);    -   c. the first sequence is comprised by an essential gene (or RNA        thereof) and the second sequence is comprised by an essential or        virulence gene (or RNA thereof); or    -   d. the first sequence is comprised by a virulence gene (or RNA        thereof) and the second sequence is comprised by an essential or        virulence gene (or RNA thereof).

Each vector may be as described above, eg, a phage capable of infectinga host cell or conjugative plasmid capable of introduction into a hostcell. In an example, the vectors are in combination with an antibioticagent (eg, a beta-lactam antibiotic).

Each first cell (host cell) may be a Staphylococcus, Streptococcus,Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio or Clostridiumhost cell. In an example, each first cell (host cell) is a Firmicutescell, eg, a Staphylococcus, Streptococcus, Listeria or Clostridium cell.

In an example, each engineered nucleic acid sequence comprises asequence R1-S1-R1′ for expression and production of the respective crRNA(eg, comprised by a single guide RNA) in the host cell, (i) wherein R1is a first CRISPR repeat, R1′ is a second CRISPR repeat, and R1 or R1′is optional; and (ii) S1 is a first CRISPR spacer that comprises orconsists of a nucleotide sequence that is 95% or more identical to saidtarget sequence.

In an example, R1 and R1′ are at least 95% identical respectively to thefirst and second repeat sequences of a CRISPR array of the host cellspecies. In an example, R1 and R1′ are at least 95% (eg, 96, 97, 98, 99or 100%) identical respectively to the first (5′-most) and second (therepeat immediately 3′ of the first repeat) repeat sequences of a CRISPRarray of said species, eg, of a said host cell of said species. In anexample, R1 and R1′ are functional with a Type II Cas9 nuclease (eg, a Sthermophilus, S pyogenes or S aureus Cas9) or Type I Cas3 to modify thetarget in a said host cell.

In one aspect, the method involves the following use, as demonstrated bythe worked experimental Example:

The use of wild-type endogenous Cas nuclease activity of the first cell(host cell) population to inhibit growth of the population, wherein eachhost cell has an endogenous CRISPR/Cas system having wild-type Casnuclease activity, the use comprising transforming host cells of thepopulation, wherein each transformed host cell is transformed with anengineered nucleotide sequence for providing host modifying (HM) cRNA orguide RNA (gRNA) in the host cell, the HM-cRNA or gRNA comprising asequence that is capable of hybridising to a host cell targetprotospacer sequence for guiding endogenous Cas to the target, whereinthe cRNA or gRNA is cognate to an endogenous Cas nuclease of the hostcell that has said wild-type nuclease activity and followingtransformation of the host cells growth of the population is inhibited.

By “cognate to” it is intended that the endogenous Cas is operable withcrRNA or gRNA sequence to be guided to the target in the host cell. Theskilled addressee will understand that such Cas guiding is generally afeature of CRISPR/Cas activity in bacterial and archaeal cells, eg,wild-type CRISPR/Cas activity in bacterial or archaeal cells havingendogenous active wild-type CRISPR/Cas systems.

By “wild-type” Cas activity it is intended, as will be clear to theskilled addressee, that the endogenous Cas is not an engineered Cas orthe cell has not been engineered to de-repress the endogenous Casactivity. This is in contrast to certain bacteria where Cas nucleaseactivity is naturally repressed (ie, there is no wild-type Cas nucleaseactivity or none that is useful for the present invention, which on thecontrary is applicable to addressing wild-type host cells in situ forexample where the endogenous Cas activity can be harnessed to effectcell population growth inhibition).

In the worked Examples below, inhibition was addressed in a bacterialpopulation (a gram positive Firmicutes) on a solid surface. A >10-foldinhibition of host cell population growth was achieved. Targeting wasdirected to an antibiotic resistance gene and an essential gene. Thedemonstration of the invention's ability to inhibit host cell growth ona surface is important and desirable in embodiments where the inventionis for treating or preventing diseases or conditions mediated or causedby microbiota as disclosed herein in a human or animal subject. Suchmicrobiota are typically in contact with tissue of the subject (eg, gut,tissue) and thus we believe that the demonstration of activity toinhibit growth of a microbiota bacterial species (exemplified byStreptococcus) on a surface supports this utility. Targeting microbiotaon plant surfaces is also a desired application.

In an example, inhibition of first cell (host cell) population growth isat least 2, 3, 4, 5, 6, 7, 8, 9 or 10-fold compared to the growth ofcells of the same species or strain not exposed to said engineerednucleotide sequence. For example, growth inhibition is indicated by alower bacterial colony number of a first sample of host cells (alone orin a mixed bacterial population, eg, a microbiota or faecal sample ofthe patient after treatment) by at least 2, 3, 4, 5, 6, 7, 8, 9 or10-fold compared to the colony number of a second sample of the hostcells (alone or in a mixed bacterial population, eg, a microbiota orfaecal sample of the patient before treatment), wherein the first cellshave been transformed by said engineered nucleotide sequence but thesecond sample has not been exposed to said engineered nucleotidesequence. In an embodiment, the colony count is determined 12, 24, 36 or48 hours after the first sample has been exposed to the engineeredsequence. In an embodiment, the colonies are grown on solid agar invitro (eg, in a petri dish). It will be understood, therefore, thatgrowth inhibition can be indicated by a reduction (<100% growth comparedto no treatment, ie, control sample growth) in growth of first (host)cells or populations, or can be a complete elimination of such growth.In an example, growth of the host cell population is reduced by at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 95%, ie, over a predetermined timeperiod (eg, 24 hours or 48 hours following combination with the cRNA orgRNA, TALEN, meganuclease, zinc finger etc in the host cells), ie,growth of the host cell population is at least such percent lower thangrowth of a control host cell population that has not been exposed tosaid cRNA or gRNA etc but otherwise has been kept in the same conditionsfor the duration of said predetermined period. In an example, percentreduction of growth is determined by comparing colony number in a sampleof each population at the end of said period (eg, at a time ofmid-exponential growth phase of the control sample). For example, afterexposing the test population to the crRNA or gRNA etc at time zero, asample of the test and control populations is taken and each sample isplated on an agar plate and incubated under identical conditions forsaid predetermined period. At the end of the period, the colony numberof each sample is counted and the percentage difference (ie, test colonynumber divided by control colony number and then times by 100, and thenthe result is subtracted from 100 to give percentage growth reduction).The fold difference is calculated by dividing the control colony numberby the test colony number.

Inhibition of population growth can be indicated, therefore, by areduction in proliferation of first (host) cell number in thepopulation. This may be due to cell killing by the nuclease and/or bydownregulation of host cell proliferation (division and/or cell growth)by the action of the nuclease on the target protospacer sequence. In anembodiment of a treatment or prevention as disclosed herein, host cellburden of the human or animal subject is reduced, whereby the disease orcondition is treated (eg, reduced or eliminated) or prevented (ie, therisk of the subject developing the disease or condition) is reduced oreliminated.

In an example, wild-type host cell endogenous Cas9 or cfp1 activity isused. In an example, wild-type host cell endogenous Cas3 and/or CASCADEactivity is used. The engineered nucleotide sequence may not be incombination with an exogenous Cas nuclease-encoding sequence.Optionally, said Cas nuclease is a nickase.

In an example, the formation of bacterial colonies of said host cells isinhibited following said dysbiosis or step (b). In an example,proliferation of host cells is inhibited following said dysbiosis orstep (b). In an example, host cells are killed following said dysbiosisor step (b).

In another aspect, the method comprises producing ex vivo a medicamentfor administration to the patient for causing said dysbiosis or step (b)for treating or preventing the disease or condition, wherein themedicament comprises a modified mixed bacterial population (eg, obtainedfrom faeces or gut microbiota of one or more human donors or saidpatient), wherein the modified population is administered to the patientto cause said dysbiosis or in step (b) to alter the balance of speciesor strains in the patient's gut microbiota, thereby altering theproportion of the first cells in the gut microbiota. The modified mixedpopulation can be produced ex vivo using guided nuclease modificationtechniques as described herein. Thus, for example, the method can beused to reduce the proportion of a specific Firmicutes sub-populationand spare Bacteroidetes in the mixed population, eg, for producing amedicament for treating or preventing a metabolic or GI condition ordisease disclosed herein. In this way, the invention can use a modifiedbacterial transplant (eg, a modified faecal transplant) medicament forsuch use or for said treatment or prevention in a human or animal. Forexample, the method can be used to modify one or more microbiota invitro to produce a modified collection of bacteria for administration toa human or animal for medical use (eg, treatment or prevention of ametabolic condition (such as obesity or diabetes) or a GI tractcondition (eg, any such condition mentioned herein) or a cancer (eg, aGI tract cancer). In an example, the transformation of bacterial cellswith phage or plasmid vectors comprising engineered nucleic acidsequences as described herein is carried out in vitro, or the engineerednucleotide sequence is comprised by nucleic acid that is electroporatedinto host cells. In an example, the nucleic acid are RNA (eg, copies ofthe gRNA). In another example, the nucleic acid are DNA encoding thecrRNA or gRNA for expression thereof in host cells.

Thus, in an example, the invention provides an engineered nucleotidesequence for providing host cell modifying (HM) cRNA or guide RNA (gRNA)in a population of wild-type bacterial host cells comprised by amicrobiota of a plant, yeast, environmental, soil, human or animalsubject for use in the method of the invention, the cRNA or gRNAcomprising a sequence that is capable of hybridising to a host celltarget protospacer sequence for guiding Cas to the target, wherein thecRNA or gRNA is cognate to an endogenous host cell Cas nuclease that haswild-type nuclease activity, wherein following transformation of hostcells growth of the population is inhibited and the disease or conditionis treated or prevented, or the therapy or treatment is modulated.

In an example, the engineered nucleotide sequence comprises a HM-CRISPRarray. In an example, the engineered nucleotide sequence encodes asingle guide RNA. In an example, the engineered nucleotide sequence is aguide RNA (eg, a single guide RNA) or crRNA. In an example, theengineered sequence is comprised by a bacteriophage that is capable ofinfecting the host cells, wherein the transformation comprisestransduction of the host cells by the bacteriophage. The bacteriophagecan be a phage as described herein. In an example, the engineerednucleotide sequence is comprised by a plasmid (eg, a conjugativeplasmid) that is capable of transforming host cells. The plasmid can bea plasmid as described herein. In an example, the engineered nucleotidesequence is comprised by a transposon that is capable of transfer intoand/or between host cells. The transposon can be a transposon asdescribed herein.

Any method of the invention can comprise transforming host cells withnucleic acid vectors for producing cRNA or gRNA in the cells. Forexample, the vectors or nucleic acid comprising the engineerednucleotide sequence are administered orally, intravenously, topically,ocularly, intranasally, by inhalation, by rectal administration, or byany other route of administration disclosed herein or otherwise to thepatient, wherein the administration transforms the first (host) cellswith the vectors or nucleic acid.

In an example, the first are mixed with second bacteria in themicrobiota of the patient or subject. Optionally, the second bacteriaspecies is E coli, L lactis or S thermophilus, as shown in the workedExample below, such are strains that co-exist symbiotically in human andanimal gut microbiota. The Example also addresses targeting in a mixedgram positive and gram negative bacterial population. Additionally, theExample addresses a population of Firmicutes (S thermophilus) and apopulation of Enterobacteriaceae (E coli), both of which are found inhuman microbiota. Other examples of Enterobacteriaceae are Salmonella,Yersinia pestis, Klebsiella, Shigella, Proteus, Enterobacter, Serratia,and Citrobacter.

In an example, the condition or disease is a metabolic orgastrointestinal disease or condition, eg, obesity, IBD, IBS, Crohn'sdisease or ulcerative colitis. In an example, the condition or diseaseis a cancer, eg, a solid tumour or a GI cancer (eg, stomach cancer),liver cancer or pancreatic cancer. In an example, the condition isresistance or reduced responsiveness to an antibiotic (eg, anyantibiotic disclosed herein).

In an example, each first (host) cell comprises an endogenous RNase IIIthat is operable with the engineered sequence in the production of saidHM-crRNA in the cell. In an alternative, one or more of the vectorscomprises a nucleotide sequence encoding such a RNase III for expressionof the RNase III in the host cell.

In an example, the essential gene (comprising the target) encodes a DNApolymerase of the cell. This is exemplified below.

In an example, the cRNA or gRNA comprises a sequence that is capable ofhybridising to a host cell target protospacer sequence that is aadjacent a NGG, NAG, NGA, NGC, NGGNG, NNGRRT or NNAGAAW protospaceradjacent motif (PAM), eg, a AAAGAAA or TAAGAAA PAM (these sequences arewritten 5′ to 3′). In an embodiment, the PAM is immediately adjacent the3′ end of the protospacer sequence. In an example, the Cas is a Saureus, S theromophilus or S pyogenes Cas. In an example, the Cas isCpf1 and/or the PAM is TTN or CTA.

In an example, each engineered nucleotide sequence or vector comprises asaid CRISPR array or a sequence encoding a said crRNA or gRNA andfurther comprises an antibiotic resistance gene (eg, kanamycinresistance), wherein the HM-crRNA or gRNA does not target the antibioticresistance gene. In an example, the target sequence is comprised by anantibiotic resistance gene of the host cell, wherein the antibiotic isdifferent from the first antibiotic (eg, kanamycin). In this way, theengineered sequence or vector is able to target the host withouttargeting itself. By exposing the host cells to the first antibiotic(eg, by orally or intravenously administering it to the patient), onecan promote retention of the engineered sequence or vector therein bypositive selection pressure since cells containing the first antibioticresistance gene will have a survival advantage in the presence of thefirst antibiotic (when host cells that are not transformed by theengineered sequence or vectors are not resistant to the firstantibiotic). Thus, an example provides: The method of the inventioncomprising exposing the first (host) cell population to said antibiotic(eg, kanamycin) and said engineered sequence or vector(s), for promotingmaintenance of cRNA or gRNA-encoding sequences in host cells; or theengineered sequence, array or vector of the invention is in combinationwith said antibiotic.

In an example the sequence encoding the cRNA or gRNA is under aconstitutive promoter (eg, a strong promoter) operable in the host cellspecies, or an inducible promoter.

In an example, the or each first (host) cell is a gram positive cell. Inanother example, the or each first (host) cell is a gram positive cell.

The invention also provides: An ex vivo mixed population of microbiotabacteria obtainable by the method by isolation of a gut microbiotasample from the patient after carrying out the method, or by isolationof a faecal sample of the patient after carrying out the method. In anexample, the mixed population is in a container for medical ornutiritional use. For example, the container is a sterilised container,eg, an inhaler, intranasal delivery device or connected to a syringe orIV needle. In an example, the mixed population is useful foradministration to a human or animal to populate a microbiome thereof fortreating a disease or condition (eg, a disease or condition disclosedherein).

Herein, in an example the Bacteroides is a species selected from caccae,capillosus, cellulosilyticus, coprocola, coprophilus, coprosuis,distasonis, dorei, eggerthii, faecis, finegoldii, fluxus, fragalis,intestinalis, melaninogenicus, nordii, oleiciplenus, oralis, ovatus,pectinophilus, plebeius, stercoris, thetaiotaomicron, uniformis,vulgatus and xylanisolvens. For example, the Bacteroides isthetaiotaomicron. In an example, the first (host cell) population orsecond bacteria comprise a plurality of different Bacteroidetes species,or a plurality of Bacteroides species (eg, comprising B thetaiotaomicronand B fragalis), or Bacteroides and Prevotella species. Herein, in anexample, the Prevotella is a species selected from bergensis, bivia,buccae, buccalis, copri, melaninogenica, oris, ruminicola, tannerae,timonensis and veroralis. In an alternative, the first (host) cells orsecond bacteria are Firmicutes cells, for example comprising orconsisting of one or more Firmicutes selected from Anaerotruncus,Acetanaerobacterium, Acetitomaculum, Acetivibrio, Anaerococcus,Anaerofilum, Anaerosinus, Anaerostipes, Anaerovorax, Butyrivibrio,Clostridium, Capracoccus, Dehalobacter, Dialister, Dorea, Enterococcus,Ethanoligenens, Faecalibacterium, Fusobacterium, Gracilibacter,Guggenheimella, Hespellia, Lachnobacterium, Lachnospira, Lactobacillus,Leuconostoc, Megamonas, Moryella, Mitsuokella, Oribacterium, Oxobacter,Papillibacter, Proprionispira, Pseudobutyrivibrio, Pseudoramibacter,Roseburia, Ruminococcus, Sarcina, Seinonella, Shuttleworthia,Sporobacter, Sporobacterium, Streptococcus, Subdoligranulum,Syntrophococcus, Thermobacillus, Turibacter and Weisella. In an example,the first (host) cells or second bacteria comprise or consist ofClostridium (eg, dificile) cells (and optionally the othersub-population consists of Bacteroides (eg, thetaiotaomicron) cells). Inan example, the first (host) cells or second bacteria comprise orconsist of Enterococcus cells (and optionally the other cells consist ofBacteroides (eg, thetaiotaomicron) cells). In an example, the first(host) cells or second bacteria comprise or consist of Ruminococcuscells (and optionally the other cells consist of Bacteroides (eg,thetaiotaomicron) cells). In an example, the first (host) cells orsecond bacteria comprise or consist of Streptococcus cells (andoptionally the other cells consist of Bacteroides (eg, thetaiotaomicron)cells). In an example, the first (host) cells or second bacteriacomprise or consist of Faecalibacterium cells (and optionally the othercells consist of Bacteroides (eg, thetaiotaomicron) cells). For example,the Faecalibacterium is a Faecalibacterium prausnitzii (eg, A2-165,L2-6, M21/2 or SL3/3).

In an example, the first (host) cells or second bacteria consist ofStreptococcus cells (optionally S thermophilus and/or pyogenes cells)and the second bacteria consists of Bacteroides (eg, thetaiotaomicron)and/or Enterobacteriaceae (eg, E coli) cells.

In an example, the first (host) cells are infectious disease pathogensof humans, an animal (eg, non-human animal) or a plant.

In an example, the first (host) cells are selected from a species ofEscherichia (eg, E coli O157:H7 or O104: H4), Shigella (eg,dysenteriae), Salmonella (eg, typhi or enterica, eg, serotypetyphimurium, eg, DT 104), Erwinia, Yersinia (eg, pestis), Bacillus,Vibrio, Legionella (eg, pneumophilia), Pseudomonas (eg, aeruginosa),Neisseria (eg, gonnorrhoea or meningitidis), Bordetella (eg, pertussus),Helicobacter (eg, pylori), Listeria (eg, monocytogenes), Agrobacterium,Staphylococcus (eg, aureus, eg, MRSA), Streptococcus (eg, pyogenes orthermophilus), Enterococcus, Clostridium (eg, dificile or botulinum),Corynebacterium (eg, amycolatum), Mycobacterium (eg, tuberculosis),Treponema, Borrelia (eg, burgdorferi), Francisella, Brucella,Campylobacter (eg, jejuni), Klebsiella (eg, pneumoniae), Frankia,Bartonella, Rickettsia, Shewanella, Serratia, Enterobacter, Proteus,Providencia, Brochothrix, Bifidobacterium, Brevibacterium,Propionibacterium, Lactococcus, Lactobacillus, Pediococcus, Leuconostoc,Vibrio (eg, cholera, eg, O139, or vulnificus), Haemophilus (eg,influenzae), Brucella (eg, abortus), Franciscella, Xanthomonas, Erlichia(eg, chaffeensis), Chlamydia (eg, pneumoniae), Parachlamydia,Enterococcus (eg, faecalis or faceim, eg, linezolid-resistant),Oenococcus and Acinetoebacter (eg, baumannii, eg, multiple drugresistant).

In an example, the first (host) cells are Staphylococcus aureus cells,eg, resistant to an antibiotic selected from methicillin,vancomycin-resistant and teicoplanin.

In an example, the first (host) cells are Pseudomonas aeuroginosa cells,eg, resistant to an antibiotic selected from cephalosporins (eg,ceftazidime), carbapenems (eg, imipenem or meropenem), fluoroquinolones,aminoglycosides (eg, gentamicin or tobramycin) and colistin.

In an example, the first (host) cells are Klebsiella (eg, pneumoniae)cells, eg, resistant to carbapenem.

In an example, the first (host) cells are Streptoccocus (eg, pneumoniaeor pyogenes) cells, eg, resistant to an antibiotic selected fromerythromycin, clindamycin, beta-lactam, macrolide, amoxicillin,azithromycin and penicillin.

In an example, the first (host) cells are Salmonella (eg, serotypeTyphi) cells, eg, resistant to an antibiotic selected from ceftriaxone,azithromycin and ciprofloxacin.

In an example, the first (host) cells are Shigella cells, eg, resistantto an antibiotic selected from ciprofloxacin and azithromycin.

In an example, the first (host) cells are Mycobacterium tuberculosiscells, eg, resistant to an antibiotic selected from Resistance toisoniazid (INH), rifampicin (RMP), fluoroquinolone, amikacin, kanamycinand capreomycin.

In an example, the first (host) cells are Enterococcus cells, eg,resistant to vancomycin.

The method of claim 13, wherein all of the host cells areEnterobacteriaceae cells, eg, resistant to an antibiotic selected from acephalosporin and carbapenem.

In an example, the first (host) cells are E. coli cells, eg, resistantto an antibiotic selected from trimethoprim, itrofurantoin, cefalexinand amoxicillin.

In an example, the first (host) cells are Clostridium (eg, dificile)cells, eg, resistant to an antibiotic selected from fluoroquinoloneantibiotic and carbapenem.

In an example, the first (host) cells are Neisseria gonnorrhoea cells,eg, resistant to an antibiotic selected from cefixime (eg, an oralcephalosporin), ceftriaxone (an injectable cephalosporin), azithromycinand tetracycline.

In an example, the first (host) cells are Acinetoebacter baumanniicells, eg, resistant to an antibiotic selected from beta-lactam,meropenem and a carbapenem.

In an example, the first (host) cells are Campylobacter cells, eg,resistant to an antibiotic selected from ciprofloxacin and azithromycin.

In an example, the second species or strain is a human gut commensalspecies or strain and/or a human gut probiotic species or strain.

In an example, the second species or strain is a Bacteroidetes (eg,Bacteroides) and optionally the host cells are gram positive bacterialcells.

In an example, the first cells are Firmicutes cells.

In an example, causing said dysbiosis or step (b) is carried out bytargeting the sub-population of first cells by administering thereto ananti-bacterial or anti-archaeal agent simultaneously or sequentiallywith said immune cell population, whereby first cells are killed or thesub-population growth is reduced, thereby reducing the proportion ofsaid sub-population in the gut microbiota of the patient.

In an example, the method reduces first (host) cell population growth byat least 5, 10, 20, 50 or 100-fold compared to the growth of a controlpopulation of host cells that have not received said guided nuclease(eg, Cas) modification.

In an example, the method inhibits host cell population growth on a gutsurface.

In an example, for each host cell the system comprises componentsaccording to (i) to (iv):—

(i) at least one nucleic acid sequence encoding a Cas nuclease;

(ii) an engineered HM-CRISPR array comprising a spacer sequence andrepeats encoding a HM-crRNA, the HM-crRNA comprising a sequence thathybridises to a host cell target sequence to guide said Cas to thetarget in the host cell to modify the target sequence;(iii) an optional tracrRNA sequence or a DNA sequence expressing atracrRNA sequence;(iv) wherein said components of the system are split between the hostcell and at least one nucleic acid vector that transforms the host cell,whereby the HM-crRNA guides Cas to the target to modify the host targetsequence in the host cell; and wherein the target sequence is modifiedby the Cas whereby the host cell is killed or host cell growth isreduced;the method comprising introducing the vectors of (iv) into host cellsand expressing said HM-crRNA in the host cells, allowing HM-cRNA tohybridise to host cell target sequences to guide Cas to the targets inthe host cells to modify target sequences, whereby host cells are killedor host cell growth is reduced, thereby altering the proportion of saidsub-population in the microbiota.

In an example, component (i) is endogenous to each host cell.

In an example, each vector is a virus or phage.

In an example, each target sequence is adjacent a NNAGAAW or NGGNGprotospacer adjacent motif (PAM).

In an example, alternatively HM-crRNA and tracrRNA are comprised by asingle guide RNA (gRNA), the method comprising introducing said gRNAinto host cells or expressing the gRNA in host cells.

In an example, the microbiota comprises a second bacterial or archaealspecies, wherein each of the first and second species is a respectivespecies of the same phylum (eg, both Firmicutes species) and the growthof the second bacteria is not inhibited by the HM-system; or wherein themicrobiota comprises a second bacterial or archaeal strain, wherein eachof the first and second bacteria or archaea is a respective strain ofthe same species and the growth of the second bacteria or archaea is notinhibited by the HM-system.

In an example, the microbiota comprises a second bacterial species,wherein each of the first and second species is a respectivegram-positive species and the growth of the second bacteria is notinhibited by the HM-system.

In an example, each target sequence is comprised by an antibioticresistance gene, virulence gene or essential gene of the host cell.

In an example, each first cell is a Staphylococcus, Streptococcus,Pseudomonas, Salmonella, Listeria, E coli, Desulfovibrio, Vibrio orClostridium cell.

In an example, the dysbiosis or step (b) comprises stimulating Panethcells of the patient by gut Bacteroides (eg, B thetaiotamicron), whereinthe altered microbiota produced by the dysbiosis or step (b) comprisesan increased proportion of Bacteroides first bacteria compared with themicrobiota before the dysbiosis or step (b), whereby Paneth cells arestimulated and the cell therapy is modulated.

Bacteroides can affect expression of Paneth cell proteins. Smallintestinal crypts house stem cells that serve to constantly replenishepithelial cells that die and are lost from the villi. Paneth cells(immune systems cells similar to neutrophils), located adjacent to thesestem cells, protect them against microbes by secreting a number ofantimicrobial molecules (defensins) into the lumen of the crypt, and itis possible that their protective effect even extends to the maturecells that have migrated onto the villi. In animal models, B.thetaiotaomicron can stimulate production of an antibiotic Paneth cellprotein (Ang4) that can kill certain pathogenic organisms (e.g.,Listeria monocytogenes). Listeria monocytogenes is a strong T_(H)1 cellinducer, and thus by stimulating Paneth cells in certain aspects of theinvention, this may be useful to skew immunity from TH1 to other celltypes, such as T_(H)17. This may be useful for increasing or enhancingthe immune cell therapy of the invention. For example, this may beuseful when the invention comprises administering T_(H)17-based celltherapy (eg, CAR-T_(H)17 cells) to the patient.

In an example, the dysbiosis or step (b) comprises developing an immuneresponse in the patient to gut Bacteroides (eg, B thetaiotamicron),wherein the altered microbiota produced by the dysbiosis or step (b)comprises an increased proportion of Bacteroides first bacteria comparedwith the microbiota before the dysbiosis or step (b), whereby the celltherapy is modulated.

In an example, the dysbiosis or step (b) comprises altering the relativeproportion of a or said sub-population of first cells in the gutmicrobiota of the patient, thereby producing an altered gut microbiotathat modulates the immune cell therapy in the patient, wherein thedysbiosis or step (b) comprises killing first cells of saidsub-population or inhibiting growth of said sub-population by usingguided nuclease targeting to the genome of first cells comprised by thesub-population.

The invention also provides: A bacterial or archaeal transplant foradministration to a patient for therapy of a disease or condition in thepatient using the method of the invention. Optionally the transplantcomprises cells of said first species. Optionally the transplantcomprises cells of said second species (and eg, does not comprise cellsof the first species).

The invention also provides: A bacterial or archaeal transplant foradministration to a subject (eg, a plant or yeast) or to soil or anenvironment for modulating a treatment thereof using the method of theinvention. Optionally the transplant comprises cells of said firstspecies. Optionally the transplant comprises cells of said secondspecies (and eg, does not comprise cells of the first species).

In an example, the yeast comprises a population of yeast cells of thesame species or strain. For example, the yeast is comprised by themicrobiota, eg, the yeast comprises a sub-population of cells of themicrobiota, such as where the targeted cells are bacteria or archaeacells also comprised by the microbiota. In an embodiment, the methodkills the targeted cells (eg, bacteria) or reduces growth of the targetcell sub-population comprised by the microbiota, wherein release of (orthe concentration of) one or more chemicals or messengers by targetcells in the microbiota is reduced. For example, the one or morechemicals or messengers that mediate quorum sensing in microbes (eg,bacteria) of the microbiota is reduced. This may be useful to shape thegrowth of target cell and/or other microbial cell populations in themicrobiota. In an example, the one or more chemicals or messengersinhibit growth of the yeast or kill the yeast, and thus reduction of therelease or concentration of the chemical or messenger(s) in themicrobiota is advantageous to promote growth and/or maintenance of theyeast in the microbiota. In an embodiment of these examples, thetreatment is nutrition of the yeast or treatment of the yeast with agrowth promoter (eg, wherein the yeast are for foodstuff or beverageproduction, or for production of a biotechnology or medical product,such as an antibody eg, the yeast is Saccharaomyces). In anotherexample, the chemical or messenger(s) promote yeast growth, wherein theyeast treatment is treatment with a yeast killing agent (eg, anfungicide). This is useful to promote efficacy of the killing treatmentwhen the yeast are undesirable (eg, an undesirable mould). In an examplewhen the subject is a plant, the one or more chemicals or messengersinhibit growth of the plant or kill the plant, and thus reduction of therelease or concentration of the chemical or messenger(s) in themicrobiota is advantageous to promote growth of the plant and/or inhibitkilling of the plant. In an embodiment of these examples, the treatmentis nutrition of the plant or treatment of the yeast with a fertilizer ornitrogen fixing agent. In an embodiment, the treatment is a pesticidetreatment of the plant, such as a treatment that is inhibited or reducedin the presence of the targeted microbiota cells.

In an example, the plant is a crop plant (eg, wheat, barley, cereal orlivestock fodder plant), fruit plant (eg, apple, orange, citrus, lime,lemon, raspberry, strawberry, berry or banana plant), legume plant,sugar cane plant, spice plant, garden plant, vegetable plant, grass,tree or flowering plant. For example, the plant is a tuber plant, eg, apotato or a sweet potato (eg, and the first, host or targeted bacteriaare Pectobacterium atrosepticum cells, optionally wherein the treatmentis storage (eg, cold storage), washing, a pesticide or herbicidetreatment, fertilising or hydrating of the plant of a crop thereof (eg,a potato crop)). For example, the plant is a tobacco plant (eg, and thefirst, host or targeted bacteria are Ralstonia solanacearum cells,optionally wherein the treatment is storage (eg, cold storage), washing,a pesticide or herbicide treatment, fertilising or hydrating of theplant of a crop thereof (eg, a tobacco leaf crop)).

In an example, the subject is a protozoa. In an example, the subject orpatient is a fish. In an example, the subject or patient is a bird. Inan example, the subject or patient is a reptile. In an example, thesubject or patient is an arachnid. In an example, the subject is a yeastcell (eg, a Saccharomyces cell). In an example, the subject or patientis an animal (eg, a rodent, mouse or rat). In an example, the subject orpatient is a human (eg, an embryo or not an embryo). In an example, thesubject or patient is a companion pet animal (eg, a bird, horse, dog,cat or rabbit). In an example, the subject or patient is an insect (aninsect at any stage of its lifecycle, eg, egg, larva or pupa, eg, a flyor crawling insect or a beetle). In an example, the subject or patientis a cephalopod or nematode. In an example, the subject or patient is aplant or animal pest. In an example, the treatment of an animal or humanmay be a nutritional treatment, therapy of a disease or condition,prophylais of a disease or condition, ocular treatment, pesticidetreatment, dental treatment, topical treatment or digestion treatment.In an example, the method enhances immunity of the subject or patientagainst a pathogen (eg a pathogenic parasite, protozoan, virus orbacteria). In an example, the treatment or therapy is a combinationtreatment or therapy practiced on the human or animal, wherein the humanor animal is administered a first medicament in combination with asecond medicament or radiation. The first and/or second medicament maybe an antibody therapy or immune cell transplant (eg, CAR-T or TILstransplant) therapy (and the immune modulation aspect of the inventionmay be advantageous for modulating such therapies). In an example, eachmedicament or treatment is selected from Table 2.

In an example, the disease or condition is diabetes (eg, Type I or IIdiabetes, insulin-resistant diabetes (eg, insulin-resistant Type IIdiabetes), onset of insulin resistance in a diabetic or pre-diabeticpatient or reduction in insulin responsiveness in a diabetic orpre-diabetic patient. Optionally additionally in this example, the firstor host cells that are targeted are Prevotella copri or Bacteroidesvulgatus cells. In an embodiment, both Prevotella copri and Bacteroidesvulgatus cells are targeted (ie, killed and/or population growthreduced, or reduced in the microbiota following administration of atransplant as described herein) in the patient, wherein said disease orcondition is treated or prevented.

The invention also provides: the HM-CRISPR/Cas system, HM-array orHM-crRNA for administration to a patient for therapy of a disease orcondition in the patient using the method of the invention.

The invention also provides: A kit comprising an ex vivo population ofimmune cells for adoptive cell therapy of a patient, wherein the kitfurther comprises the transplant, system, array or crRNA, optionallywherein the immune cells are selected from CAR-T cells, T-cellsexpressing engineered T-cell receptors (TCRs), tumour infiltratinglymphocytes (TILs) and NK cells.

Mobile Genetic Elements (Mges)

In an example, each vector is a nucleic acid vector comprising orconsisting of a mobile genetic element (MGE), wherein the MGE comprisesan origin of transfer (oriT) and a CRISPR array for modifying a targetsequence of the genome of a host cell or the genome of a virus (eg,prophage) in a host cell. Examples of MGEs are ICEs, transposons,plasmids and bacteriophage. An origin of transfer (oriT) is a shortsequence (eg, up to 500 bp) that is necessary for transfer of the DNAthat contains it from a bacterial host to recipient during conjugation.The oriT is cis-acting—it is found on the same DNA that is beingtransferred, and it is transferred along with the DNA. A typical originof transfer comprises three functionally defined domains: a nickingdomain, a transfer domain, and a termination domain.

Reference is made to the ICEberg database(http://db-mml.sjtu.edu.cn/ICEberg/), which provides examples ofsuitable ICEs for the invention and sources for suitable oriT. In anexample, the ICE is a member of an ICE family comprising an ICE selectedfrom the group 1 to 28, or the oriT is an oriT of a member of such afamily: 1=SXT/R391; 2=Tn916; 3=Tn4371; 4=CTnDOT/ERL; 5=ICEcic; 6=ICEBs1;7=ICEHin1056; 8=PAPI-1; 9=ICEM1Sym(R7A); 10=ICESt1; 11=SPI-7;12=ICE6013; 13=ICEKp1; 14=TnGBS1; 15=Tn5253; 16=ICESa2603; 17=ICEYe1;18=10270-RD.2; 19=Tn1207.3; 20=Tn1806; 21=ICEA5632; 22=ICEF-I/II;23=ICEAPG2; 24=ICEM; 25=10270-RD.1; 26=Tn5801; 27=PPI-1; 28=ICEF-III.Family descriptions are found in the ICEberg database. For example, theTn916 family was defined by Roberts et al (2009) (Trends Microbiol. 2009June; 17(6):251-8. doi: 10.1016/j.tim.2009.03.002. Epub 2009 May 20; “Amodular master on the move: the Tn916 family of mobile geneticelements”, Roberts A, Mullany P). Elements belonging to the Tn916 familyare defined by the following criteria: they must have the generalorganization shown in Roberts et al, and they must have a core region(conjugation and regulation module) that is similar in sequence andstructure to the original Tn916 at the DNA level. Exceptions are someconjugative transposons, such as Tn1549 which have been previouslyclassified in this family and those with a high degree of proteinsimilarity as described in corresponding references. Optionally, the ICEis a transposon, eg, a conjugative transposon. In an example, the MGE isa mobilisable transposon that is mobilisable in the presence of afunctional helper element, optionally wherein the transposon is incombination with a said helper element.

Optionally the vector is a plasmid, optionally wherein the MGE is atransposon comprised by the plasmid. For example, the transposon is aconjugative transposon. In an example the transposon is a mobilisabletransposon (eg, mobilisable using one or more factors encoded by theplasmid, eg, by genes outside the transposon sequence of the plasmid).Optionally, the transposon is a Type I transposon. Optionally, thetransposon is a Type II transposon. Optionally, the vector oriT is anoriT of a Bacteroidetes (eg, Bacteroidales or Bacteroides) or Prevotellatransposon. This useful when the first (host) cells are Bacteroidetes(eg, Bacteroidales or Bacteroides) or Prevotella respectively.Optionally, the vector oriT is a CTnDot, CTnERL SXT/R391, Tn916 orTn4371 family transposon oriT.

Optionally, the method comprises exposing the patient's microbiota to avector or MGE (eg, one described above) that comprises a toxin-antioxinmodule that comprises an anti-toxin gene that is operable in the secondbacteria, but is not operable or has reduced operation in first (host)cells. Thus, first cells are killed and second bacteria are spared,thereby altering the proportion of first cells in the patient'smicrobiota.

Split Crispr/Cas System

In one aspect, endogenous Cas of the first (host) cells is harnessed andoperates with the engineered sequences comprised by vectors (eg, phage)that are introduced into host cells. This aspect is advantageous to freeup space in vectors, for example viruses or phage that have restrictedcapacity for carrying exogenous sequence. By freeing up space, one isable to include more targeting spacers or arrays, which is useful forevading host resistance. It is advantageous, for example to harness theendogenous Cas endonuclease rather than encode it in thevector—especially for bulky Cas sequences such as sp or saCas9.Additionally, there is not chance of inferior compatibility as may beseen with some exogenous Cas from non-host sources. The ability toreduce virus, eg, phage genome size, may also be beneficial forpromoting host cell uptake (infection and/or maintenance of the virus inhost cells). In some examples, an advantage is that invasion of the hostby the vector (eg, phage) may upregulate host CRISPR/Cas activity,including increased expression of host Cas nucleases—in an attempt ofthe host to combat invading nucleic acid. This, however, is also usefulto provide endogenous Cas for use with the invention when these use cRNAor gRNA that are recognised by the host Cas. In the case where theinvention involves cRNA or gRNA targeting a host CRISPR array, this thenpromotes inactivation of the host CRISPR array itself, akin to a“suicidal” host cell which then uses its own Cas nuclease to inactivateits own CRISPR systems.

Thus, the vectors may lack a Cas nuclease (eg, aCas9)-encoding sequence.

Optionally, the endogenous first (host) cell system is a CRISPR/Cas9system. Optionally, the nuclease is a Type I Cas nuclease. Optionally,the nuclease is a Type II Cas nuclease (eg, a Cas9). Optionally, thenuclease is a Type III Cas nuclease.

To save even more space, optionally a tracrRNA sequence is not providedby the vectors, but is a tracrRNA sequence of an endogenous host cellCRISPR/Cas system, wherein the tracrRNA is capable of hybridising withthe HM-crRNA in the cell for subsequent processing into mature crRNA forguiding Cas to the target in the host cell.

Generally Applicable Features

The following features apply to any configuration (eg, in any of itsaspects, embodiments, concepts, paragraphs or examples) of theinvention:—

In an example, the target sequence is a chromosomal sequence, anendogenous host cell sequence, a wild-type host cell sequence, anon-viral chromosomal host cell sequence and/or a non-phage sequence(ie, one more or all of these), eg, the sequence is a wild-type hostchromosomal cell sequence such as an antibiotic resistance gene oressential gene sequence comprised by a host cell chromosome. In anexample, the sequence is a host cell plasmid sequence, eg, an antibioticresistance gene sequence.

In an example, at least two target sequences are modified by Cas, forexample an antibiotic resistance gene and an essential gene. Multipletargeting in this way may be useful to reduce evolution of escape mutanthost cells.

In an example, the Cas is a wild-type endogenous host cell Cas nuclease.In an example, each host cell has constitutive Cas nuclease activity,eg, constitutive wild-type Cas nuclease activity. In an example, thehost cell is a bacterial cell; in another example the host cell is anarchael cell. Use of an endogenous Cas is advantageous as this enablesspace to be freed in vectors cRNA or gRNA. For example, Type II Cas9nucleotide sequence is large and the use of endogenous Cas of the hostcell instead is advantageous in that instance when a Type II CRISPR/Cassystem is used for host cell modification in the present invention. Themost commonly employed Cas9, measuring in at 4.2 kilobases (kb), comesfrom S pyogenes. While it is an efficient nuclease, the molecule'slength pushes the limit of how much genetic material a vector canaccommodate, creating a barrier to using CRISPR in the tissues of livinganimals and other settings described herein (see F. A. Ran et al., “Invivo genome editing using Staphylococcus aureus Cas9,” Nature,doi:10.1038/nature14299, 2015). Thus, in an embodiment, the vector ofthe invention is a AAV vector or has an exogenous DNA insertion capacityno more than an AAV vector, and the Cas is an endogenous Cas of the hostcell, wherein the cell is a bacterial or archaeal cell. S thermophilusCas9 (UniProtKB-G3ECR1 (CAS9_STRTR)) nucleotide sequence has a size of1.4 kb.

In an example, the vector is a viral vector. Viral vectors have aparticularly limited capacity for exogenous DNA insertion, thus viruspackaging capacity needs to be considered. Room needs to be left forsequences encoding vital viral functions, such as for expressing coatproteins and polymerase. In an example, the vector is a phage vector oran AAV or lentiviral vector. Phage vectors are useful where the host isa bacterial cell.

By use of the term “engineered” it will be readily apparent to theskilled addressee that the array, sequence, vector, cRNA, gRNA, MGE orany other configuration, concept, aspect, embodiment, paragraph orexample etc of the invention is non-naturally occurring. For example,the MGE, vector, sequence or array comprises one or more sequences orcomponents not naturally found together with other sequences orcomponents of the MGE, vector, sequence or array. For example, the arrayor sequence is recombinant, artificial, synthetic or exogenous (ie,non-endogenous or not wild-type) to the or each host cell.

In an example, the array or sequence of the invention is an engineeredversion of an array or sequence isolated, for example isolated from ahost cell. In an example, the engineered array or sequence is not incombination with a Cas endonuclease-encoding sequence that is naturallyfound in a cell.

Studies suggest that Bacteroides have a role in preventing infectionwith Clostridium difficile. The development of the immune response thatlimits entry and proliferation of potential pathogens is profoundlydependent upon B fragilis. Also, Paneth cell proteins may produceantibacterial peptides in response to stimulation by B thetaiotomicron,and these molecules may prevent pathogens from colonizing the gut. Inaddition, B thetaiotomicron can induce Paneth cells to produce abactericidal lectin, RegIII, which exerts its antimicrobial effect bybinding to the peptidoglycan of gram-positive organisms. Thus, the useof the invention in any of its configurations for increasing theproportion of Bacteroides (eg, thetaiotomicron and/or fragalis) in thepatient's microbiota is useful for limiting pathogenic bacterialcolonisation of the population or a gut of a human or non-human animal.

Hooper et al demonstrated that B thetaiotomicron can modify intestinalfucosylation in a complex interaction mediated by a fucose repressorgene and a signaling system. Using transcriptional analysis it wasdemonstrated that B thetaiotaomicron can modulate expression of avariety of host genes, including those involved in nutrient absorption,mucosal barrier fortification, and production of angiogenic factors.

Optionally, the host (or first and/or second bacteria) is a gramnegative bacterium (eg, a spirilla or vibrio). Optionally, the host (orfirst and/or second bacteria) is a gram positive bacterium. Optionally,the host (or first and/or second bacteria) is a mycoplasma, chlamydiae,spirochete or mycobacterium. Optionally, the host (or first and/orsecond bacteria) is a Streptococcus (eg, pyogenes or thermophilus) host.Optionally, the host (or first and/or second bacteria) is aStaphylococcus (eg, aureus, eg, MRSA) host. Optionally, the host (orfirst and/or second bacteria) is an E. coli (eg, O157: H7) host, eg,wherein the Cas is encoded by the vecor or an endogenous host Casnuclease activity is de-repressed. Optionally, the host (or first and/orsecond bacteria) is a Pseudomonas (eg, aeruginosa) host. Optionally, thehost (or first and/or second bacteria) is a Vibro (eg, cholerae (eg,O139) or vulnificus) host. Optionally, the host (or first and/or secondbacteria) is a Neisseria (eg, gonnorrhoeae or meningitidis) host.Optionally, the host (or first and/or second bacteria) is a Bordetella(eg, pertussis) host. Optionally, the host (or first and/or secondbacteria) is a Haemophilus (eg, influenzae) host. Optionally, the host(or first and/or second bacteria) is a Shigella (eg, dysenteriae) host.Optionally, the host (or first and/or second bacteria) is a Brucella(eg, abortus) host. Optionally, the host (or first and/or secondbacteria) is a Francisella host. Optionally, the host (or first and/orsecond bacteria) is a Xanthomonas host. Optionally, the host (or firstand/or second bacteria) is a Agrobacterium host. Optionally, the host(or first and/or second bacteria) is a Erwinia host. Optionally, thehost (or first and/or second bacteria) is a Legionella (eg, pneumophila)host. Optionally, the host (or first and/or second bacteria) is aListeria (eg, monocytogenes) host. Optionally, the host (or first and/orsecond bacteria) is a Campylobacter (eg, jejuni) host. Optionally, thehost (or first and/or second bacteria) is a Yersinia (eg, pestis) host.Optionally, the host (or first and/or second bacteria) is a Borelia (eg,burgdorferi) host. Optionally, the host (or first and/or secondbacteria) is a Helicobacter (eg, pylori) host. Optionally, the host (orfirst and/or second bacteria) is a Clostridium (eg, dificile orbotulinum) host. Optionally, the host (or first and/or second bacteria)is a Erlichia (eg, chaffeensis) host. Optionally, the host (or firstand/or second bacteria) is a Salmonella (eg, typhi or enterica, eg,serotype typhimurium, eg, DT 104) host. Optionally, the host (or firstand/or second bacteria) is a Chlamydia (eg, pneumoniae) host.Optionally, the host (or first and/or second bacteria) is aParachlamydia host. Optionally, the host (or first and/or secondbacteria) is a Corynebacterium (eg, amycolatum) host. Optionally, thehost (or first and/or second bacteria) is a Klebsiella (eg, pneumoniae)host. Optionally, the host (or first and/or second bacteria) is aEnterococcus (eg, faecalis or faecim, eg, linezolid-resistant) host.Optionally, the host (or first and/or second bacteria) is aAcinetobacter (eg, baumannii, eg, multiple drug resistant) host.

A tracrRNA sequence may be omitted from a array or vector of theinvention, for example for Cas systems of a Type that does not usetracrRNA.

In an example, the Cas guided to the target is an exonuclease.Optionally a nickase as mentioned herein is a double nickase. An exampleof a nickase is a Cas9 nickase, ie, a Cas9 that has one of the twonuclease domains inactivated—either the RuvC and/or HNH domain.

Mention herein of using vector DNA can also in an alternative embodimentapply mutatis mutandis to vector RNA where the context allows. Forexample, where the vector is an RNA vector. All features of theinvention are therefore in the alternative disclosed and to be read as“RNA” instead of “DNA” when referring to vector DNA herein when thecontext allows. In an example, the or each vector also encodes a reversetranscriptase.

In an example, the or each array or engineered nucleotide sequence isprovided by a nanoparticle vector or in liposomes.

In an example, the Cas is a Cas nuclease for cutting, dead Cas (dCas)for interrupting or a dCas conjugated to a transcription activator foractivating the target.

In an example, the or each array or engineered sequence comprises anexogenous promoter functional for transcription of the crRNA or gRNA inthe host.

In an embodiment the array or engineered sequence is contained in avirophage vector and the host is alternatively a virus which can infecta cell. For example, the host is a large virus that may have infected anamoeba cell. For example, the host is a Sputnik virus, Pithovirus,mimivirus, mamavirus, Megavirus or Pandoravirus, eg, wherein the hostvirus is in water. In an example of this embodiment, the invention isfor water or sewage treatment (eg, purification, eg, waterway, river,lake, pond or sea treatment).

In an embodiment the or each vector or engineered sequence is or iscomprised by a ΦNM1 phage, eg, wherein the host cell(s) is a S. aureus(eg, MRSA) cell.

For example the method is practiced on a mammalian subject, eg, a human,rodent, mouse or rat. For example the method is practiced on avertebrate, reptile, bird or fish.

The cell population can be administered to the patient in one or moredoses. For example, the method comprises administering an antibacterialagent to cause said dysbiosis, or administering a bacterial transplantto the patient to cause said dysbiosis.

Wherein the method reduces the cell therapy, the therapy can bedownregulated, dampened or switched off, eg, to reduce or prevent anunwanted side-effect of the cell therapy (eg, a CAR-T therapy sideeffect in a human patient, such as CRS). Wherein the method increasesthe cell therapy, the therapy can be upregulated, enhance or switchedon, eg, to enhance cytotoxicity against one target cells.

The method treats or prevents (ie, reduces the risk of) the disease orcondition. This may be complete or partial treatment or prevention, ie,a reduction but not complete reduction of the disease/condition orsymptoms thereof; or a reducing of the risk but not total prevention ofthe disease/condition or a symptom thereof. Similarly, the method treatsor prevents (ie, reduces the risk of) an undesirable symptom of thedisease or condition or the therapy (eg, CRS).

Concepts:

-   1. A method of modulating an adoptive immune cell therapy of a    disease or condition in a patient, the method comprising    -   a. Carrying out adoptive immune cell therapy in the patient,        comprising administering a population of immune cells to the        patient, wherein administration of said immune cells is capable        of treating the disease or condition in the patient; and    -   b. Causing bacterial microbiota (eg, gut microbiota) dysbiosis        in the patient, whereby said dysbiosis modulates the immune cell        therapy in the patient.-   2. A method of modulating an adoptive immune cell therapy of a    disease or condition in a patient, the method comprising    -   a. Carrying out adoptive immune cell therapy in the patient,        comprising administering a population of immune cells to the        patient, wherein administration of said immune cells is capable        of treating the disease or condition in the patient; and    -   b. Altering the relative proportion of a sub-population of cells        of a first bacterial species or strain, or archaeal species or        strain, in a microbiota (eg, gut microbiota) of the patient,        thereby producing an altered microbiota that modulates the        immune cell therapy in the patient.-   3. The method of concept 2, wherein step (b) is carried out by    targeting the sub-population of first cells by administering thereto    an anti-bacterial or anti-archaeal agent simultaneously or    sequentially with said immune cell population, whereby first cells    are killed or the sub-population growth is reduced, thereby reducing    the proportion of said sub-population in the microbiota of the    patient.-   4. The method of any preceding concept, wherein the cell therapy is    an adoptive T-cell therapy.-   5. The method of concept 4, wherein cells selected from the group    consisting of CD4⁺ T-cells, CD8⁺ T-cells, T_(H)1 cells and T_(H)17    cells are administered to the patient in step (a).-   6. The method of concept 4 or 5, wherein T_(H)17 cells are modulated    in the patient.-   7. The method of concept 4, 5 or 6, wherein T_(reg) cells are    modulated in the patient.-   8. The method of any preceding concept, wherein the cell therapy is    enhanced.-   9. The method of any preceding concept wherein T_(H)17 cells are    upregulated in the patient and/or T_(reg) cells are downregulated in    the patient.-   10. The method of any preceding concept, wherein CD4⁺ T-cells are    upregulated in the patient.-   11. The method of any preceding concept, wherein CD8⁺ T-cells are    upregulated in the patient.-   12. The method of any preceding concept, wherein one or more of    central memory T cells (T_(CM)), effector memory T cells (T_(EM)),    stem cell memory cells (T_(SCM)) and effector cells (T_(eff)) are    upregulated in the patient, wherein the cells are comprised by the    immune cell population administered in step (a) and/or are progeny    thereof.-   13. The method of any one of concepts 1 to 7, wherein the cell    therapy is reduced.-   14. The method of concept 13, wherein the method reduces or prevents    the risk of cytokine release syndrome (CRS) in the patient.-   15. The method of any one of concepts 1 to 7, 13 and 14, wherein,    T_(H)17 cells are downregulated in the patient and/or T_(reg) cells    are upregulated in the patient.-   16. The method of any one of concepts 1 to 7 and 13 to 15, wherein    CD4⁺ T-cells are downregulated in the patient.-   17. The method of any one of concepts 1 to 7 and 13 to 16, wherein    CD8⁺ T-cells are downregulated in the patient.-   18. The method of any one of concepts 1 to 7 and 13 to 17, wherein    one or more of central memory T cells (T_(CM)), effector memory T    cells (T_(EM)), stem cell memory cells (T_(SCM)) and effector cells    (T_(eff)) are downregulated in the patient, wherein the cells are    comprised by the immune cell population administered in step (a)    and/or are progeny thereof.-   19. The method of any preceding concept, wherein the immune cell    population comprises CAR-T cells and/or T-cells expressing    engineered T-cell receptors (TCRs) and/or tumour infiltrating    lymphocytes (TILs).-   20. The method of any preceding concept, wherein the immune cell    population comprises engineered autologous or allogeneic immune    cells, eg, T-cells, NK cells and/or TILs.-   21. The method of concept 19 or 20, wherein the T-cells are CD4⁺    T-cells or T_(H)17 cells.-   22. The method of any preceding concept, wherein step (b) increases    the proportion of Bacteroides (eg, B fragalis and/or B    thetaiotamicron) in the microbiota.-   23. The method of any one of concepts 1 to 21, wherein step (b)    decreases the proportion of Bacteroides (eg, B fragalis and/or B    thetaiotamicron) in the microbiota.-   24. The method of any preceding concept, wherein step (b) increases    the proportion of Bacteroidetes to Firmicutes in the microbiota.-   25. The method of any one of concepts 1 to 23, wherein step (b)    decreases the proportion of Bacteroidetes to Firmicutes in the    microbiota.-   26. The method of any preceding concept, wherein step (b) reduces    the proportion of one or more Clostridium species or strain (eg,    wherein each species is a cluster IV or XIVa Clostridium species) in    the microbiota.-   27. The method of any one of concepts 1 to 25, wherein step (b)    increases the proportion of one or more Clostridium species or    strain (eg, wherein each species is a cluster IV or XIVa Clostridium    species) in the microbiota.-   28. The method of any preceding concept, wherein step (b) reduces    the proportion of Bifidobacterium (eg, B bifidum) in the microbiota.-   29. The method of any one of concepts 1 to 27, wherein step (b)    increases the proportion of Bifidobacterium (eg, B bifidum) in the    microbiota.-   30. The method of any preceding concept, wherein step (b) comprises    altering the relative proportion of a or said sub-population of    first cells in the microbiota of the patient, thereby producing an    altered microbiota that modulates the immune cell therapy in the    patient, wherein the sub-population comprises host cells of said    first species or strain, the method comprising    -   a. combining the microbiota with multiple copies of engineered        nucleic acid sequences encoding host modifying (HM) crRNAs, and    -   b. expressing HM-crRNAs in host cells, wherein each engineered        nucleic acid sequence is operable with a Cas nuclease in a        respective host cell to form a HM-CRISPR/Cas system and the        engineered sequence comprises        -   (iii) spacer and repeat sequences encoding a HM-crRNA;        -   (iv) the HM-crRNA comprising a sequence that is capable of            hybridizing to a host cell target sequence to guide Cas            nuclease to the target sequence in the host cell; and    -   optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -   whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said sub-population in the microbiota.-   31. The method of concept 30, comprising using endogenous Cas    nuclease of host cells for modification of target nucleotide    sequences.-   32. The method of concept 30 or 31, comprising reducing host cell    population growth by at least 5-fold compared to the growth of a    control population of host cells that have not received said Cas    modification.-   33. The method of any one of concepts 30 to 32, comprising    inhibiting host cell population growth on a gut surface.-   34. The method of any one of concepts 30 to 33, wherein the    microbiota comprises cells of a second bacterial species or strain,    or archaeal species or strain, wherein the second species or strain    has a 16s ribosomal RNA-encoding DNA sequence that is at least 80%    identical to an 16s ribosomal RNA-encoding DNA sequence of the host    cell species or strain, wherein the growth of the second cells in    the microbiota is not inhibited by said HM-system.-   35. The method of concept 34, wherein the second species or strain    is a human gut commensal species or strain and/or a human gut    probiotic species or strain.-   36. The method of concept 34 or 35, wherein the second species or    strain is a Bacteroidetes (eg, Bacteroides) and optionally the host    cells are gram positive bacterial cells.-   37. The method of any one of concepts 30 to 36, wherein the first    cells are Firmicutes cells.-   38. The method of any one of concepts 30 to 37, wherein for each    host cell the system comprises components according to (i) to (iv):—    -   (i) at least one nucleic acid sequence encoding a Cas nuclease;    -   (ii) an engineered HM-CRISPR array comprising a spacer sequence        and repeats encoding a HM-crRNA, the HM-crRNA comprising a        sequence that hybridises to a host cell target sequence to guide        said Cas to the target in the host cell to modify the target        sequence;    -   (iii) an optional tracrRNA sequence or a DNA sequence expressing        a tracrRNA sequence;    -   (iv) wherein said components of the system are split between the        host cell and at least one nucleic acid vector that transforms        the host cell, whereby the HM-crRNA guides Cas to the target to        modify the host target sequence in the host cell; and    -   wherein the target sequence is modified by the Cas whereby the        host cell is killed or host cell growth is reduced;    -   the method comprising introducing the vectors of (iv) into host        cells and expressing said HM-crRNA in the host cells, allowing        HM-cRNA to hybridise to host cell target sequences to guide Cas        to the targets in the host cells to modify target sequences,        whereby host cells are killed or host cell growth is reduced,        thereby altering the proportion of said sub-population in the        microbiota.-   39. The method of concept 38, wherein component (i) is endogenous to    each host cell.-   40. The method of concept 38 or 39, wherein each vector is a virus    or phage.-   41. The method of any one of concepts 30 to 40, wherein each target    sequence is adjacent a NNAGAAW or NGGNG protospacer adjacent motif    (PAM).-   42. The method of any one of concepts 30 to 41, wherein    alternatively HM-crRNA and tracrRNA are comprised by a single guide    RNA (gRNA), the method comprising introducing said gRNA into host    cells or expressing the gRNA in host cells.-   43. The method of any one of concepts 30 to 35 and 37 to 42, wherein    the microbiota comprises a second bacterial or archaeal species,    wherein each of the first and second species is a respective species    of the same phylum (eg, both Firmicutes species) and the growth of    the second bacteria is not inhibited by the HM-system; or wherein    the microbiota comprises a second bacterial or archaeal strain,    wherein each of the first and second bacteria or archaea is a    respective strain of the same species and the growth of the second    bacteria or archaea is not inhibited by the HM-system.-   44. The method of any one of concepts 30 to 43, wherein the    microbiota comprises a second bacterial species, wherein each of the    first and second species is a respective gram-positive species and    the growth of the second bacteria is not inhibited by the HM-system.-   45. The method of any one of concepts 30 to 44, wherein each target    sequence is comprised by an antibiotic resistance gene, virulence    gene or essential gene of the host cell.-   46. The method of any preceding concept, wherein each first cell is    a Staphylococcus, Streptococcus, Pseudomonas, Salmonella, Listeria,    E coli, Desulfovibrio, Vibrio or Clostridium cell.-   47. The method of any preceding concept, wherein step (b) comprises    stimulating Paneth cells of the patient by gut Bacteroides (eg, B    thetaiotamicron), wherein the altered microbiota produced by    step (b) comprises an increased proportion of Bacteroides first    bacteria compared with the microbiota before step (b), whereby    Paneth cells are stimulated and the cell therapy is modulated.-   48. The method of any preceding concept, wherein step (b) comprises    developing an immune response in the patient to gut Bacteroides (eg,    B thetaiotamicron), wherein the altered microbiota produced by    step (b) comprises an increased proportion of Bacteroides first    bacteria compared with the microbiota before step (b), whereby the    cell therapy is modulated.-   49. The method of any preceding concept, wherein step (b) comprises    altering the relative proportion of a or said sub-population of    first cells in the microbiota of the patient, thereby producing an    altered microbiota that modulates the immune cell therapy in the    patient, wherein step (b) comprises killing first cells of said    sub-population or inhibiting growth of said sub-population by using    guided nuclease targeting to the genome of first cells comprised by    the sub-population.-   50. A bacterial or archaeal transplant for administration to a    patient for therapy of a disease or condition in the patient using    the method of any preceding concept, optionally wherein the    transplant comprises cells of said first species.-   51. A HM-CRISPR/Cas system, HM-array or HM-crRNA as recited in any    one of concepts 30 to 45 for administration to a patient for therapy    of a disease or condition in the patient using the method of any one    of concepts 1 to 49.-   52. A kit comprising an ex vivo population of immune cells for    adoptive cell therapy of a patient, wherein the kit further    comprises a transplant, system, array or crRNA of concept 50 or 51,    optionally wherein the immune cells are selected from CAR-T cells,    T-cells expressing engineered T-cell receptors (TCRs), tumour    infiltrating lymphocytes (TILs) and NK cells.

Aspects:

-   -   1. An ex vivo population of immune cells for use in a method of        adoptive cell therapy of a patient for treating or preventing a        disease or condition in the patient, the method comprising        -   a. Carrying out adoptive immune cell therapy in the patient,            comprising administering cells of said population to the            patient, wherein administration of said immune cells is            capable of treating the disease or condition in the patient;            and        -   b. Causing gut bacterial microbiota dysbiosis in the            patient, whereby said dysbiosis modulates the immune cell            therapy in the patient and said disease or condition is            treated or prevented.    -   2. An ex vivo population of immune cells for use in a method of        adoptive cell therapy of a patient for treating or preventing a        disease or condition in the patient, the method comprising        -   a. Carrying out adoptive immune cell therapy in the patient,            comprising administering cells of said population to the            patient, wherein administration of said immune cells is            capable of treating the disease or condition in the patient;            and        -   b. Altering the relative proportion of a sub-population of            cells of a first bacterial species or strain, or archaeal            species or strain, in the gut microbiota of the patient,            thereby producing an altered gut microbiota that modulates            the immune cell therapy in the patient.    -   3. The immune cell population of Aspect 2, wherein step (b) is        carried out by targeting the sub-population of first cells by        administering thereto an anti-bacterial or anti-archaeal agent        (eg, a guided nuclease) simultaneously or sequentially with said        immune cell population, whereby first cells are killed or the        sub-population growth is reduced, thereby reducing the        proportion of said sub-population in the gut microbiota of the        patient.    -   4. The immune cell population of any preceding Aspect wherein        the cell therapy is an adoptive T-cell therapy.    -   5. The immune cell population of Aspect 4, wherein cells        selected from the group consisting of CD4⁺ T-cells, CD8⁺        T-cells, T_(H)1 cells and T_(H)17 cells are administered to the        patient in step (a).    -   6. The immune cell population of Aspect 4 or 5, wherein T₁ 17        cells are modulated in the patient.    -   7. The immune cell population of Aspect 4, 5 or 6, wherein        T_(reg) cells are modulated in the patient.    -   8. The immune cell population of any preceding Aspect, wherein        the cell therapy is enhanced.    -   9. The hrimune cell population of any preceding Aspect wherein        T_(H)17 cells are upregulated in the patient and/or T_(reg)        cells are downregulated in the patient.    -   10. The immune cell population of any preceding Aspect, wherein        CD4⁺ T-cells re upregulated in the patient.    -   11 The immune cell population of any preceding Aspect, wherein        CD8⁺ T-cells are upregulated in the patient.    -   12, The immune cell population of any preceding Aspect, wherein        one or more of central memory T cells (T_(CM)), effector memory        T cells (T_(EM)), stem cell memory cells (T_(SCM)) and effector        cells (T_(eff)) are upregulated in the patient, wherein the        cells are comprised by the immune cell population administered        in step (a) and/or are progeny thereof.    -   13. The immune cell population of any one of Aspects 1 to 7,        wherein the cell therapy is reduced.    -   14. The immune cell population of Aspect 13, wherein the method        reduces or prevents the risk of cytokine release syndrome (CRS)        in the patient.    -   15, The immune cell population of any one of Aspects 1 to 7, 13        and 14, wherein, T_(H)17 cells are downregulated in the patient        and/or T_(reg) cells are upregulated in the patient.    -   16. The immune cell population of any one of Aspects 1 to 7 and        13 to 15, wherein CD4⁺ T-cells are downregulated in the patient.    -   17. The immune cell population of any one of Aspects 1 to 7 and        13 to 16, wherein CD8+ T cells are downregulated in the patient.    -   18. The immune cell population of any one of Aspects 1 to 7 and        13 to 17, wherein one or more of central memory T cells        (T_(CM)), effector memory T cells (T_(EM)), stem cell memory        cells (T_(SCM)) and effector cells (T_(eff)) are downregulated        in the patient, wherein the cells are comprised by the immune        cell population administered in step (a) and/or are progeny        thereof.    -   19. The immune cell population of any preceding Aspect, wherein        the immune cell population comprises or consists of CAR-T cells        and/or T-cells expressing engineered T-cell receptors (TCRs)        and/or tumour infiltrating lymphocytes (TILs).    -   20. The immune cell population of any preceding Aspect, wherein        the immune cell population comprises or consists of engineered        autologous or allogeneic immune cells, eg, T-cells, NK cells        and/or TILs.    -   21. The immune cell population of Aspect 19 or 20, wherein the        T-cells are CD4⁺ T-cells or T_(H)17 cells.    -   22. The immune cell population of any preceding Aspect, wherein        step (b) increases the proportion of Bacteroides (eg, B fragalis        and/or B thetaiotamicron) in the gut microbiota.    -   23. The immune cell population of any one of Aspects 1 to 21,        wherein step (b) decreases the proportion of Bacteroides (eg, B        fragalis and/or B thetaiotamicron) in the gut microbiota.    -   24. The immune cell population of any preceding Aspect, wherein        step (b) increases the proportion of Bacteroidetes to Firmicutes        in the gut microbiota.    -   25. The immune cell population of any one of Aspects 1 to 23,        wherein step (b) decreases the proportion of Bacteroidetes to        Firmicutes in the gut microbiota.    -   26. The immune cell population of any preceding Aspect, wherein        step (b) reduces the proportion of one or more Clostridium        species or strain (eg, wherein each species is a cluster IV or        XIVa Clostridium species) in the gut microbiota.    -   27. The immune cell population of any one of Aspects 1 to 25,        wherein step (b) increases the proportion of one or more        Clostridium species or strain (eg, wherein each species is a        cluster IV or XIVa Clostridium species) in the gut microbiota.    -   28. The immune cell population of any preceding Aspect, wherein        step (b) reduces the proportion of Bifidobacterium (eg, B        bifidum) in the gut microbiota.    -   29. The immune cell population of any one of Aspects 1 to 27,        wherein step (b) increases the proportion of Bifidobacterium        (eg, B bifidum) in the gut microbiota.    -   30. The immune cell population of any preceding Aspect, wherein        step (b) comprises altering the relative proportion of a or said        sub-population of first cells in the gut microbiota of the        patient, thereby producing an altered gut microbiota that        modulates the immune cell therapy in the patient, wherein the        sub-population comprises host cells of said first species or        strain, the method comprising        -   a. combining the microbiota with multiple copies of            engineered nucleic acid sequences encoding host modifying            (HM) crRNAs, and        -   b. expressing HM-crRNAs in host cells, wherein each            engineered nucleic acid sequence is operable with a Cas            nuclease in a respective host cell to form a HM-CRISPR/Cas            system and the engineered sequence comprises        -   (i) spacer and repeat sequences encoding a HM-crRNA;        -   (ii) the HM-crRNA comprising a sequence that is capable of            hybridizing to a host cell target sequence to guide Cas            nuclease to the target sequence in the host cell; and    -   optionally the HM-system comprises a tracrRNA sequence or a DNA        sequence expressing a tracrRNA sequence;    -   whereby HM-crRNAs guide Cas modification of host target        sequences in host cells, whereby host cells are killed or the        host cell population growth is reduced, thereby reducing the        proportion of said sub-population in the microbiota.    -   31. The immune cell population of Aspect 30, comprising using        endogenous Cas nuclease of host cells for modification of target        nucleotide sequences.    -   32. The immune cell population of Aspect 30 or 31, comprising        reducing host cell population growth by at least 5-fold compared        to the growth of a control population of host cells that have        not received said Cas modification.    -   33. The immune cell population of any one of Aspects 30 to 32,        comprising inhibiting host cell population growth on a gut        surface.    -   34. The immune cell population of any one of Aspects 30 to 33,        wherein the microbiota comprises cells of a second bacterial        species or strain, or archaeal species or strain, wherein the        second species or strain has a 16s ribosomal RNA-encoding DNA        sequence that is at least 80% identical to an 16s ribosomal        RNA-encoding DNA sequence of the host cell species or strain,        wherein the growth of the second cells in the microbiota is not        inhibited by said HM-system.    -   35. The immune cell population of Aspect 34, wherein the second        species or strain is a human gut commensal species or strain        and/or a human gut probiotic species or strain.    -   36. The immune cell population of Aspect 34 or 35, wherein the        second species or strain is a Bacteroidetes (eg, Bacteroides)        and optionally the host cells are gram positive bacterial cells.    -   37. The immune cell population of any one of Aspects 30 to 36,        wherein the first cells are Firmicutes cells.    -   38. The immune cell population of any one of Aspects 30 to 37,        wherein for each host cell the system comprises components        according to (i) to (iv):—        -   (i) at least one nucleic acid sequence encoding a Cas            nuclease;        -   (ii) an engineered HM-CRISPR array comprising a spacer            sequence and repeats encoding a HM-crRNA, the HM-crRNA            comprising a sequence that hybridises to a host cell target            sequence to guide said Cas to the target in the host cell to            modify the target sequence;        -   (iii) an optional tracrRNA sequence or a DNA sequence            expressing a tracrRNA sequence;        -   (iv) wherein said components of the system are split between            the host cell and at least one nucleic acid vector that            transforms the host cell, whereby the HM-crRNA guides Cas to            the target to modify the host target sequence in the host            cell; and        -   wherein the target sequence is modified by the Cas whereby            the host cell is killed or host cell growth is reduced;        -   the method comprising introducing the vectors of (iv) into            host cells and expressing said HM-crRNA in the host cells,            allowing HM-cRNA to hybridise to host cell target sequences            to guide Cas to the targets in the host cells to modify            target sequences, whereby host cells are killed or host cell            growth is reduced, thereby altering the proportion of said            sub-population in the microbiota.    -   39. The immune cell population of Aspect 38, wherein        component (i) is endogenous to each host cell.    -   40. The immune cell population of Aspect 38 or 39, wherein each        vector is a virus or phage.    -   41. The immune cell population of any one of Aspects 30 to 40,        wherein each target sequence is adjacent a NNAGAAW or NGGNG        protospacer adjacent motif (PAM).    -   42. The immune cell population of any one of Aspects 30 to 41,        wherein alternatively HM-crRNA and tracrRNA are comprised by a        single guide RNA (gRNA), the method comprising introducing said        gRNA into host cells or expressing the gRNA in host cells.    -   43. The immune cell population of any one of Aspects 30 to 35        and 37 to 42, wherein the microbiota comprises a second        bacterial or archaeal species, wherein each of the first and        second species is a respective species of the same phylum (eg,        both Firmicutes species) and the growth of the second bacteria        is not inhibited by the HM-system; or wherein the microbiota        comprises a second bacterial or archaeal strain, wherein each of        the first and second bacteria or archaea is a respective strain        of the same species and the growth of the second bacteria or        archaea is not inhibited by the HM-system.    -   44. The immune cell population of any one of Aspects 30 to 43,        wherein the microbiota comprises a second bacterial species,        wherein each of the first and second species is a respective        gram-positive species and the growth of the second bacteria is        not inhibited by the HM-system.    -   45. The immune cell population of any one of Aspects 30 to 44,        wherein each target sequence is comprised by an antibiotic        resistance gene, virulence gene or essential gene of the host        cell.    -   46. The method of any preceding Aspect, wherein each first cell        is a Staphylococcus, Streptococcus, Pseudomonas, Salmonella,        Listeria, E coli, Desulfovibrio, Vibrio or Clostridium cell.    -   47. The immune cell population of any preceding Aspect, wherein        step (b) comprises stimulating Paneth cells of the patient by        gut Bacteroides (eg, B thetaiotamicron), wherein the altered        microbiota produced by step (b) comprises an increased        proportion of Bacteroides first bacteria compared with the        microbiota before step (b), whereby Paneth cells are stimulated        and the cell therapy is modulated.    -   48. The immune cell population of any preceding Aspect, wherein        step (b) comprises developing an immune response in the patient        to gut Bacteroides (eg, B thetaiotamicron), wherein the altered        microbiota produced by step (b) comprises an increased        proportion of Bacteroides first bacteria compared with the        microbiota before step (b), whereby the cell therapy is        modulated.    -   49. The immune cell population of any preceding Aspect, wherein        step (b) comprises altering the relative proportion of a or said        sub-population of first cells in the gut microbiota of the        patient, thereby producing an altered gut microbiota that        modulates the immune cell therapy in the patient, wherein        step (b) comprises killing first cells of said sub-population or        inhibiting growth of said sub-population by using guided        nuclease targeting to the genome of first cells comprised by the        sub-population.    -   50. A bacterial or archaeal transplant for administration to a        patient for therapy of a disease or condition in the patient        using the method recited in any preceding Aspect, optionally        wherein the transplant comprises cells of said first species.    -   51. A HM-CRISPR/Cas system, HM-array or HM-crRNA as recited in        any one of Aspects 30 to 45 for administration to a patient for        therapy of a disease or condition in the patient using the        method recited in any one of Aspects 1 to 49.    -   52. A kit comprising an ex vivo population of immune cells        according to any one of Aspects 1 to 49 for adoptive cell        therapy of a patient to treat said disease or condition, wherein        the kit further comprises a transplant, system, array or crRNA        of Aspect 50 or 51, optionally wherein the immune cells are        selected from CAR-T cells, T-cells expressing engineered T-cell        receptors (TCRs), tumour infiltrating lymphocytes (TILs) and NK        cells.

Optionally, the host cell(s), first cell(s), second cell(s) or mixedbacterial population is comprised by a human or a non-human animalsubject, eg, the population is comprised by a gut microbiota, skinmicrobiota, oral cavity microbiota, throat microbiota, hair microbiota,armpit microbiota, vaginal microbiota, rectal microbiota, analmicrobiota, ocular microbiota, nasal microbiota, tongue microbiota, lungmicrobiota, liver microbiota, kidney microbiota, genital microbiota,penile microbiota, scrotal microbiota, mammary gland microbiota, earmicrobiota, urethra microbiota, labial microbiota, organ microbiota ordental microbiota. Optionally, the mixed bacterial population iscomprised by a plant (eg, a tobacco, crop plant, fruit plant, vegetableplant or tobacco, eg on the surface of a plant or contained in a plant)or by an environment (eg, soil or water or a waterway or acqueousliquid).

Optionally, the disease or condition of a human or animal subject orpatient is selected from

-   -   (a) A neurodegenerative disease or condition;    -   (b) A brain disease or condition;    -   (c) A CNS disease or condition;    -   (d) Memory loss or impairment;    -   (e) A heart or cardiovascular disease or condition, eg, heart        attack, stroke or atrial fibrillation;    -   (f) A liver disease or condition;    -   (g) A kidney disease or condition, eg, chronic kidney disease        (CKD);    -   (h) A pancreas disease or condition;    -   (i) A lung disease or condition, eg, cystic fibrosis or COPD;    -   (j) A gastrointestinal disease or condition;    -   (k) A throat or oral cavity disease or condition;    -   (1) An ocular disease or condition;    -   (m) A genital disease or condition, eg, a vaginal, labial,        penile or scrotal disease or condition;    -   (n) A sexually-transmissible disease or condition, eg,        gonorrhea, HIV infection, syphilis or Chlamydia infection;    -   (o) An ear disease or condition;    -   (p) A skin disease or condition;    -   (q) A heart disease or condition;    -   (r) A nasal disease or condition    -   (s) A haematological disease or condition, eg, anaemia, eg,        anaemia of chronic disease or cancer;    -   (t) A viral infection;    -   (u) A pathogenic bacterial infection;    -   (v) A cancer;    -   (w) An autoimmune disease or condition, eg, SLE;    -   (x) An inflammatory disease or condition, eg, rheumatoid        arthritis, psoriasis, eczema, asthma, ulcerative colitis,        colitis, Crohn's disease or IBD;    -   (y) Autism;    -   (z) ADHD;    -   (aa) Bipolar disorder;    -   (bb) ALS [Amyotrophic Lateral Sclerosis];    -   (cc) Osteoarthritis;    -   (dd) A congenital or development defect or condition;    -   (ee) Miscarriage;    -   (ff) A blood clotting condition;    -   (gg) Bronchitis;    -   (hh) Dry or wet AMD;    -   (ii) Neovascularisation (eg, of a tumour or in the eye);    -   (jj) Common cold;    -   (kk) Epilepsy;    -   (ll) Fibrosis, eg, liver or lung fibrosis;    -   (mm) A fungal disease or condition, eg, thrush;    -   (nn) A metabolic disease or condition, eg, obesity, anorexia,        diabetes, Type I or Type II diabetes.    -   (oo) Ulcer(s), eg, gastric ulceration or skin ulceration;    -   (pp) Dry skin;    -   (qq) Sjogren's syndrome;    -   (rr) Cytokine storm;    -   (ss) Deafness, hearing loss or impairment;    -   (tt) Slow or fast metabolism (ie, slower or faster than average        for the weight, sex and age of the subject);    -   (uu) Conception disorder, eg, infertility or low fertility;    -   (vv) Jaundice;    -   (ww) Skin rash;    -   (xx) Kawasaki Disease;    -   (yy) Lyme Disease;    -   (zz) An allergy, eg, a nut, grass, pollen, dust mite, cat or dog        fur or dander allergy;    -   (aaa) Malaria, typhoid fever, tuberculosis or cholera;    -   (bbb) Depression;    -   (ccc) Mental retardation;    -   (ddd) Microcephaly;    -   (eee) Malnutrition;    -   (fff) Conjunctivitis;    -   (ggg) Pneumonia;    -   (hhh) Pulmonary embolism;    -   (iii) Pulmonary hypertension;    -   (jjj) A bone disorder;    -   (kkk) Sepsis or septic shock;    -   (lll) Sinusitus;    -   (mmm) Stress (eg, occupational stress);    -   (nnn) Thalassaemia, anaemia, von Willebrand Disease, or        haemophilia;    -   (ooo) Shingles or cold sore;    -   (ppp) Menstruation;    -   (qqq) Low sperm count.        Neurodegenerative or Cns Diseases or Conditions for Treatment or        Prevention by the Method

In an example, the neurodegenerative or CNS disease or condition isselected from the group consisting of Alzheimer disease, geriopsychosis,Down syndrome, Parkinson's disease, Creutzfeldt-jakob disease, diabeticneuropathy, Parkinson syndrome, Huntington's disease, Machado-Josephdisease, amyotrophic lateral sclerosis, diabetic neuropathy, andCreutzfeldt Creutzfeldt-Jakob disease. For example, the disease isAlzheimer disease. For example, the disease is Parkinson syndrome.

In an example, wherein the method of the invention is practised on ahuman or animal subject for treating a CNS or neurodegenerative diseaseor condition, the method causes downregulation of Treg cells in thesubject, thereby promoting entry of systemic monocyte-derivedmacrophages and/or Treg cells across the choroid plexus into the brainof the subject, whereby the disease or condition (eg, Alzheimer'sdisease) is treated, prevented or progression thereof is reduced. In anembodiment the method causes an increase of IFN-gamma in the CNS system(eg, in the brain and/or CSF) of the subject. In an example, the methodrestores nerve fibre and/or reduces the progression of nerve fibredamage. In an example, the method restores nerve myelin and/or reducesthe progression of nerve myelin damage. In an example, the method of theinvention treats or prevents a disease or condition disclosed inWO2015136541 and/or the method can be used with any method disclosed inWO2015136541 (the disclosure of this document is incorporated byreference herein in its entirety, eg, for providing disclosure of suchmethods, diseases, conditions and potential therapeutic agents that canbe administered to the subject for effecting treatment and/or preventionof CNS and neurodegenerative diseases and conditions, eg, agents such asimmune checkpoint inhibitors, eg, anti-PD-1, anti-PD-L1, anti-TIM3 orother antibodies disclosed therein).

Cancers for Treatment or Prevention by the Method

Cancers that may be treated include tumours that are not vascularized,or not substantially vascularized, as well as vascularized tumours. Thecancers may comprise non-solid tumours (such as haematological tumours,for example, leukaemias and lymphomas) or may comprise solid tumours.Types of cancers to be treated with the invention include, but are notlimited to, carcinoma, blastoma, and sarcoma, and certain leukaemia orlymphoid malignancies, benign and malignant tumours, and malignanciese.g., sarcomas, carcinomas, and melanomas. Adult tumours/cancers andpaediatric tumours/cancers are also included.

Haematologic cancers are cancers of the blood or bone marrow. Examplesof haematological (or haematogenous) cancers include leukaemias,including acute leukaemias (such as acute lymphocytic leukaemia, acutemyelocytic leukaemia, acute myelogenous leukaemia and myeloblasts,promyeiocytic, myelomonocytic, monocytic and erythroleukaemia), chronicleukaemias (such as chronic myelocytic (granulocytic) leukaemia, chronicmyelogenous leukaemia, and chronic lymphocytic leukaemia), polycythemiavera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent andhigh grade forms), multiple myeloma, Waldenstrom's macroglobulinemia,heavy chain disease, myeiodysplastic syndrome, hairy cell leukaemia andmyelodysplasia.

Solid tumours are abnormal masses of tissue that usually do not containcysts or liquid areas. Solid tumours can be benign or malignant.Different types of solid tumours are named for the type of cells thatform them (such as sarcomas, carcinomas, and lymphomas). Examples ofsolid tumours, such as sarcomas and carcinomas, include fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and othersarcomas, synovioma, mesothelioma, Ewing's tumour, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreaticcancer, breast cancer, lung cancers, ovarian cancer, prostate cancer,hepatocellular carcinoma, squamous eel! carcinoma, basal cell carcinoma,adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma,papillary thyroid carcinoma, pheochromocytomas sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas, medullarycarcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bileduct carcinoma, choriocarcinoma, Wilms' tumour, cervical cancer,testicular tumour, seminoma, bladder carcinoma, melanoma, and CNStumours (such as a glioma (such as brainstem glioma and mixed gliomas),glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNSlymphoma, germinoma, medu!loblastoma, Schwannoma craniopharyogioma,ependymoma, pineaioma, hemangioblastoma, acoustic neuroma,oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brainmetastases).

Autoimmune Diseases for Treatment or Prevention by the Method

-   -   Acute Disseminated Encephalomyelitis (ADEM)    -   Acute necrotizing hemorrhagic leukoencephalitis    -   Addison's disease    -   Agammaglobulinemia    -   Alopecia areata    -   Amyloidosis    -   Ankylosing spondylitis    -   Anti-GBM/Anti-TBM nephritis    -   Antiphospholipid syndrome (APS)    -   Autoimmune angioedema    -   Autoimmune aplastic anemia    -   Autoimmune dysautonomia    -   Autoimmune hepatitis    -   Autoimmune hyperlipidemia    -   Autoimmune immunodeficiency    -   Autoimmune inner ear disease (AIED)    -   Autoimmune myocarditis    -   Autoimmune oophoritis    -   Autoimmune pancreatitis    -   Autoimmune retinopathy    -   Autoimmune thrombocytopenic purpura (ATP)    -   Autoimmune thyroid disease    -   Autoimmune urticaria    -   Axonal & neuronal neuropathies    -   Balo disease    -   Behcet's disease    -   Bullous pemphigoid    -   Cardiomyopathy    -   Castleman disease    -   Celiac disease    -   Chagas disease    -   Chronic fatigue syndrome    -   Chronic inflammatory demyelinating polyneuropathy (CIDP)    -   Chronic recurrent multifocal ostomyelitis (CRMO)    -   Churg-Strauss syndrome    -   Cicatricial pemphigoid/benign mucosal pemphigoid    -   Crohn's disease    -   Cogans syndrome    -   Cold agglutinin disease    -   Congenital heart block    -   Coxsackie myocarditis    -   CREST disease    -   Essential mixed cryoglobulinemia    -   Demyelinating neuropathies    -   Dermatitis herpetiformis    -   Dermatomyositis    -   Devic's disease (neuromyelitis optica)    -   Discoid lupus    -   Dressler's syndrome    -   Endometriosis    -   Eosinophilic esophagitis    -   Eosinophilic fasciitis    -   Erythema nodosum    -   Experimental allergic encephalomyelitis    -   Evans syndrome    -   Fibromyalgia    -   Fibrosing alveolitis    -   Giant cell arteritis (temporal arteritis)    -   Giant cell myocarditis    -   Glomerulonephritis    -   Goodpasture's syndrome    -   Granulomatosis with Polyangiitis (GPA) (formerly called        Wegener's Granulomatosis)    -   Graves' disease    -   Guillain-Barre syndrome    -   Hashimoto's encephalitis    -   Hashimoto's thyroiditis    -   Hemolytic anemia    -   Henoch-Schonlein purpura    -   Herpes gestationis    -   Hypogammaglobulinemia    -   Idiopathic thrombocytopenic purpura (ITP)    -   IgA nephropathy    -   IgG4-related sclerosing disease    -   Immunoregulatory lipoproteins    -   Inclusion body myositis    -   Interstitial cystitis    -   Juvenile arthritis    -   Juvenile diabetes (Type 1 diabetes)    -   Juvenile myositis    -   Kawasaki syndrome    -   Lambert-Eaton syndrome    -   Leukocytoclastic vasculitis    -   Lichen planus    -   Lichen sclerosus    -   Ligneous conjunctivitis    -   Linear IgA disease (LAD)    -   Lupus (SLE)    -   Lyme disease, chronic    -   Meniere's disease    -   Microscopic polyangiitis    -   Mixed connective tissue disease (MCTD)    -   Mooren's ulcer    -   Mucha-Habermann disease    -   Multiple sclerosis    -   Myasthenia gravis    -   Myositis    -   Narcolepsy    -   Neuromyelitis optica (Devic's)    -   Neutropenia    -   Ocular cicatricial pemphigoid    -   Optic neuritis    -   Palindromic rheumatism    -   PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders        Associated with Streptococcus)    -   Paraneoplastic cerebellar degeneration    -   Paroxysmal nocturnal hemoglobinuria (PNH)    -   Parry Romberg syndrome    -   Parsonnage-Turner syndrome    -   Pars planitis (peripheral uveitis)    -   Pemphigus    -   Peripheral neuropathy    -   Perivenous encephalomyelitis    -   Pernicious anemia    -   POEMS syndrome    -   Polyarteritis nodosa    -   Type I, II, & III autoimmune polyglandular syndromes    -   Polymyalgia rheumatica    -   Polymyositis    -   Postmyocardial infarction syndrome    -   Postpericardiotomy syndrome    -   Progesterone dermatitis    -   Primary biliary cirrhosis    -   Primary sclerosing cholangitis    -   Psoriasis    -   Psoriatic arthritis    -   Idiopathic pulmonary fibrosis    -   Pyoderma gangrenosum    -   Pure red cell aplasia    -   Raynauds phenomenon    -   Reactive Arthritis    -   Reflex sympathetic dystrophy    -   Reiter's syndrome    -   Relapsing polychondritis    -   Restless legs syndrome    -   Retroperitoneal fibrosis    -   Rheumatic fever    -   Rheumatoid arthritis    -   Sarcoidosis    -   Schmidt syndrome    -   Scleritis    -   Scleroderma    -   Sjogren's syndrome    -   Sperm & testicular autoimmunity    -   Stiff person syndrome    -   Subacute bacterial endocarditis (SBE)    -   Susac's syndrome    -   Sympathetic ophthalmia    -   Takayasu's arteritis    -   Temporal arteritis/Giant cell arteritis    -   Thrombocytopenic purpura (TTP)    -   Tolosa-Hunt syndrome    -   Transverse myelitis    -   Type 1 diabetes    -   Ulcerative colitis    -   Undifferentiated connective tissue disease (UCTD)    -   Uveitis    -   Vasculitis    -   Vesiculobullous dermatosis    -   Vitiligo    -   Wegener's granulomatosis (now termed Granulomatosis with        Polyangiitis (GPA).        Inflammatory Diseases for Treatment or Prevention by the Method    -   Alzheimer's    -   ankylosing spondylitis    -   arthritis (osteoarthritis, rheumatoid arthritis (RA), psoriatic        arthritis)    -   asthma    -   atherosclerosis    -   Crohn's disease    -   colitis    -   dermatitis    -   diverticulitis    -   fibromyalgia    -   hepatitis    -   irritable bowel syndrome (IBS)    -   systemic lupus erythematous (SLE)    -   nephritis    -   Parkinson's disease    -   ulcerative colitis.

In an example (eg, in the method of the invention involving a mixedbacterial population), the host cell (or first cell or second cell)genus or species is selected from a genus or species listed in Table 1.In examples of the present invention, the Cas (eg, Cas nuclease such asa Type I, II or III Cas, eg, a Cas3 or 9) is a Cas comprised by bacteriaof a genus or species that is selected from a genus or species listed inTable 1, and optionally the host cell (or first cell or second cell) isof the same genus or species. In an example of this, the Cas isendogenous to said host cell (or first or second cell), which is usefulfor embodiments herein wherein endogenous Cas is used to modify a targetsequence. In this case, the HM-array may comprise one or more repeatnucleotide (eg, DNA or RNA) sequences that is at least 90, 95, 96, 97,98 or 99% identical (or is 100% identical) to a repeat sequence of saidcell, genus or species, whereby the Cas is operable with cRNA encoded bythe HM-array for modifying one or more target sequences in the cell. Inan example, the Cas is a Type I Cas3 and is used with a Type I CASCADE,wherein one or or both of the Cas3 and CASCADE are endogenous to thehost or first cells, or are vector-borne (ie, exogenous to the host orfirst cells).

In an example, the method of the invention selectively kills first cellsin the microbiota whilst not targeting second cells, eg, wherein thesecond cells are (a) of a related strain to the strain of the firstspecies or (b) of a species that is different to the first species andis phylogenetically related to the first species, wherein the secondspecies or strain has a 16s ribosomal RNA-encoding DNA sequence that isat least 80% identical to an 16s ribosomal RNA-encoding DNA sequence ofthe first cell species or strain. In an embodiment, the first cells areof a first species selected from Table 1 and the second cells are of adifferent species selected from Table 1. In an example, the species areof the same genus or are of different genera.

It will be understood that particular embodiments described herein areshown by way of illustration and not as limitations of the invention.The principal features of this invention can be employed in variousembodiments without departing from the scope of the invention. Thoseskilled in the art will recognize, or be able to ascertain using no morethan routine study, numerous equivalents to the specific proceduresdescribed herein. Such equivalents are considered to be within the scopeof this invention and are covered by the claims. All publications andpatent applications mentioned in the specification are indicative of thelevel of skill of those skilled in the art to which this inventionpertains. All publications and patent applications and all US equivalentpatent applications and patents are herein incorporated by reference tothe same extent as if each individual publication or patent applicationwas specifically and individually indicated to be incorporated byreference. The use of the word “a” or “an” when used in conjunction withthe term “comprising” in the claims and/or the specification may mean“one,” but it is also consistent with the meaning of “one or more,” “atleast one,” and “one or more than one.” The use of the term “or” in theclaims is used to mean “and/or” unless explicitly indicated to refer toalternatives only or the alternatives are mutually exclusive, althoughthe disclosure supports a definition that refers to only alternativesand “and/or.” Throughout this application, the term “about” is used toindicate that a value includes the inherent variation of error for thedevice, the method being employed to determine the value, or thevariation that exists among the study subjects.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps

The term “or combinations thereof” or similar as used herein refers toall permutations and combinations of the listed items preceding theterm. For example, “A, B, C, or combinations thereof is intended toinclude at least one of: A, B, C, AB, AC, BC, or ABC, and if order isimportant in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC,or CAB. Continuing with this example, expressly included arecombinations that contain repeats of one or more item or term, such asBB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilledartisan will understand that typically there is no limit on the numberof items or terms in any combination, unless otherwise apparent from thecontext.

Any part of this disclosure may be read in combination with any otherpart of the disclosure, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

The present invention is described in more detail in the following nonlimiting Examples.

EXAMPLES Example 1: Specific Microbiota Bacterial Population GrowthInhibition by Harnessing Wild-Type Endogenous Cas

1. Material and Methods

1.1. Strains

The following strains were used in the course of this Example andExamples 2 and 3: E. coli MG1655, E. coli TOP10, Streptococcusthermophilus LMD-9 (ATCC BAA-491, Manassas, Va.), Streptococcusthermophilus DSM 20617(T) (DSMZ, Braunschweig, Germany), Lactococcuslactis MG1363 and Streptococcus mutans Clarke 1924 DSM 20523 (DSMZ,Braunschweig, Germany).

During the course of media selection and testing of the geneticconstructs different Streptoccoci strains were used. Streptococcusthermophilus LMD-9 (ATCC BAA-491) and Escherichia coli TOP10 wereconsidered because of their compatible growth requirements. All strainswere cultivated in Todd-Hewitt broth (TH) (T1438 Sigma-Aldrich), inaerobic conditions and at 37° C., unless elsewhere indicated. Thestrains were stored in 25% glycerol at −80° C.

1.2. Differential Growth Media

All strains were grown on TH media at 37° C. for 20 hours. Selectivemedia for S. thermophilus was TH media supplemented with 3 g 1⁻¹ of2-phenylethanol (PEA). PEA was added to the media and autoclaved at 121°C. for 15 minutes at 15 psi. Agar plates were prepared by adding 1.5%(wt/vol) agar to the corresponding media. When necessary for selectionor plasmid maintenance 30 μg ml⁻¹ kanamycin was used for both S.thermophilus strains and E. coli, and 500 μg ml⁻¹ for S. mutans.

In some cases, depending on the strain and plasmid, a longer incubation,up to 48 hours, may be needed to see growth on media supplemented withPEA. In order to control for the viability of the organisms used, acontrol T_(H) agar must be done in parallel.

1.3. Cloning

E. coli (ONE SHOT® ThermoFischer TOP10 Chemically Competent cells) wasused in all subcloning procedures. PCR was carried out using Phusionpolymerase. All PCR products were purified with Nucleospin Gel and PCRClean-up by Macherey-Nagel following the manufacturer's protocol. Thepurified fragments were digested with restriction enzyme DpnI in 1× FDbuffer with 1 μl enzyme in a total volume of 34 μl. The digestedreaction was again purified with Nucleospin Gel and PCR Clean-up byMacherey-Nagel following the manufacturer's protocol. Gibson assemblywas performed in 10 μl reactions following the manufacturer's protocol(NewEngland Biolab).

Plasmid DNA was prepared using Qiagen kits according to themanufacturer's instructions. Modifications for Gram-positive strainsincluded growing bacteria in a medium supplemented with 0.5% glycine andlysozyme to facilitate cell lysis.

1.4. Transformation

1.4.1 Electro-Competent E. coli Cells and Transformation

Commercially electrocompetent cells were used for cloning and theexperiments (ONE SHOT® ThermoFischer TOP10 Chemically Competent E.coli). Electroporation was done using standard settings: 1800 V, 25 μFand 200Ω using an Electro Cell Manipulator (BTX Harvard ApparatusECM630). Following the pulse, 1 ml LB-SOC media was added and the cellswere incubated at 37° C. for 1 hour. The transformed cells were platedin LB-agar containing 50 μg ml⁻¹ of kanamycin.

1.4.2 Preparation of Electro-Competent S. thermophilus Cells

The electroporation protocol was modified from Somkuti and Steinberg,1988. An overnight culture of Streptococcus thermophilus in TH Brothsupplemented with 40 mM DL-threonine (T8375 Sigma-Aldrich) was diluted100-fold in 5 ml of the same media and grown to an OD₆₀₀ between 0.3-0.5(approximately 2.5 hours after inoculation). The cells were collected bycentrifugation at 10,000×g for 10 min at 4° C. and washed three timeswith 5 ml of ice cold wash buffer (0.5 M sucrose+10% glycerol). Afterthe cells were washed, they were suspended to an OD₆₀₀ of 15-30 inelectroporation buffer (0.5 M sucrose, 10% glycerol and 1 mM MgCl₂). Thecells in the electroporation buffer may be kept at 4° C. until use(within one hour) or aliquot 50 μl in eppendorf tubes, freezing them inliquid nitrogen and stored at −80° C. for later use.

1.4.3 Electroporation S. thermophilus Cells

1 μl of purified plasmid DNA was added to 50 μl of the cell suspensionand electroporation was carried out in 2 mm-gap electroporation cuvettespre-cooled. The electroporation setting were 2500 V, 25 μF and 200Ωusing an Electro Cell Manipulator (BTX Harvard Apparatus ECM630)Immediately after the electric pulse, 1 ml of TH broth was added to thecells and the suspension was kept on ice for 10 minutes, subsequentlythe cells were incubated for 3 h at 37° C. After allowing time forexpression of the resistance gene the cells were plated onto TH-agarplates containing 30 μg ml⁻¹ of kanamycin. Depending on the construct,colonies were visible between 12 and 48 h of incubation at 37° C.

1.5. Construction of XyIS Plasmid

All the plasmids used in this work were based on pBAV1K-T5, which is abroad-host range expression vector derived from the a cryptic plasmidpWV01 from Streptococcus cremoris (Bryksin & Matsumura, 2010), thebackbone was amplified using that contain overhangs for assembly withthe other fragments using Gibson's method.

The xylose inducible system was constructed by cloning the promoter gyrAin front of the XylR repressor (FIG. 1). The XylR repressor wasamplified from Bacillus Subtilis strain SCK6 (Zhang et al. 2011) withthe a reverse primer that includes an overhang for Gibson assembly and aforward primer, that is an ultramer used to introduce the gyrA promoter(Xie et al. 2013) and the corresponding overhang for assembly intopBAV1KT5 backbone. The resulting fragment was flanked by an mCherryamplified from pCL002 (unpublished work) with an ultramer that includePldha+PxylA hybrid promoter (Xie et al. 2013). The three resulting PCRproducts were assembled in a GIBSON MASTER MIX® (NewEngland Biolab)according to manufacturer's instructions. The product was finallytransformed in E. coli TOP10 electrocompetent cells. See FIG. 1.

1.6. Design and Construction of CRISPR Array Plasmid

Streptococcus thermophilus has 4 distinct CRISPR systems (Sapranauskas,et al. 2011), for this work the type II CRISPR1 (ST1-CRISPR) system waschosen. The design of the target sequence was based on the availablegenome sequence of LMD-9 (GenBank: CP000419.1). The ST1-CRISPR array wasdesigned to contain only the CRISPR array repeats and spacers under axylose inducible promoter (Xie et al. 2013), followed by thecorresponding tracrRNA under a strong constitutive promoter forStreptococci species (Sorg et al. 2014) (FIG. 2).

The tracrRNA plays a role in the maturation of crRNA and it is processedby S. thermophilus endogenous RNase III, forming a complex with crRNA.This complex acts as a guide for the endonuclease ST1-Cas9 (Horvath &Barrangou, 2010). After transcription of the synthetic array from thexylose inducible promoter, the endogenous Cas9 and RNAses will processit into a functional gRNA. The gRNA/Cas9 complex will cause a doublestranded break at the target location.

The design of the array used 2 specific target sequences high on GCcontent and a reduced portion of the tracrRNA (ie, a less than completetracrRNA sequence), which has been suggested not to be necessary forproper maturation of crRNA (Horvath & Barrangou, 2010).

The 2 targets were an essential gene (DNA polymerase III subunit alpha)and an antibiotic resistance gene (tetA-like gene).

Primers were used to amplify pBAV1KT5-XylR-PldhA backbone. The CRISPRarray gBlock and the backbone with overhangs were assembled in a GIBSONMASTER MIX® according to manufacturer's instructions (NewEnglandBiolabs). The product was finally transformed in E. coli TOP10electrocompetent cells.

1.7. Characterization of Xylose Inducible System in Streptoccocusthermophilus LMD-9

Overnight stationary-phase cultures were diluted 1:100 into TH brothwith corresponding antibiotic. Mid-log cells were induced with differentconcentration of D-(+)-xylose (0, 0.001, 0.01, 0.1, 0.5 and 1% wt/vol)and the cell cultures were measured either directly in medium to assessthe extent of autofluorescence of the media, on the cell suspension orthe suspension buffer (PBS buffer). 20 μl samples of the cell cultureswere diluted 1/10 on PBS buffer, on 96-well plates with flat bottoms.Fluorescence of cell suspensions or media was read on a plate reader.mCherry fluorescence was measured using an excitation wavelength of 558nm and emission at 612 nm. Absorbance of the resuspended cells wasmeasured at OD 600 nm. A minimum of three independent biologicalreplicates was done for each experiment.

1.8. Activation of CRISPR Array in S. thermophilus

S. thermophilus LMD-9 and E. coli TOP10 both with the plasmid containingthe CRISPR array targeting the DNA polymerase III and tetA of S.thermophilus were grown overnight in 3 ml cultures supplemented with 30μg ml⁻¹ of kanamycin for plasmid maintenance. The next day 96 well deepwell plates were inoculated with 500 μl of 1/100 of overnight culture infresh TH media, supplemented with 30 μg ml⁻¹ kanamycin. Mid-log cellcultures were induced with 1% xylose. The killing effect was tested onS. thermophilus and E. coli alone. For each strain and condition testeda negative control was kept without xylose. The cells were grown till˜OD 0.5 and next 10-fold serially diluted in TH media and using a96-well replicator (METTLER TOLEDO LIQUIDATOR™ 96) 5 μL volume dropswere spotted on TH agar and TH agar supplemented with g 1⁻¹ PEA plates.The plates were incubated for 24H at 37° C. and the colony forming units(CFU) were calculated from triplicate measurements.

2. Results

2.1 Growth Condition and Selective Media

We first set out to establish the bacterial strains and cultivationprotocol that would support growth for all strains we planned to use forthe co-cultivation experiments. We used S. thermophilus strain LMD-9which was able to support a similar growth as E. coli in TH broth at 37°C. (FIG. 3).

Distinguishing the different bacteria from a mixed culture is importantin order to determine cell number of the different species. WithMacConkey agar is possible to selectively grow E. coli, however there isno specific media for selective growth of S. thermophilus. PEA agar is aselective medium that is used for the isolation of gram-positive (S.thermophilus) from gram-negative (E. coli). Additionally, we found thatdifferent concentrations of PEA partially inhibit the growth of othergram positives, which allow for selection between the othergram-positive bacteria used in this work (FIG. 4). 3 g 1⁻¹ of PEA provedto selectively grow S. thermophilus LMD-9 while limiting growth of E.coli.

2.2 Design and Validation of Inducible System

An induction system for Streptococcus species was previously developedbased on the Bacillus megaterium xylose operon (FIG. 5) by creating aheterologous xylose induction cassette (Xyl-S). The xylR and xylApromoters were replaced with S. mutans' constitutively expressed gyrAand ldh promoters respectively. This expression cassette forStreptococcus species showed differences in sensitivity and expressionlevels between different species, however the system was not tested inS. thermophilus (Xie et al. 2013). Therefore we first set out tovalidate the xylose induction cassette in S. thermophilus.

An alternative version of the induction cassette was constructed by onlyreplacing the xylR promoter with the S. mutans' gyrA promoter but leftthe endogenous B. megaterium xylA promoter intact. During the design ofthe xylose inducible system we considered both versions of the induciblepromoter, the natural P_(Xyla) promoter found in Bacillus megaterium anda hybrid promoter of the highly conserved promoter P_(Idha) fused withthe repressor binding sites of P_(Xyla) promoter (FIG. 5). Only a fewStreptococcus species have been reported to metabolize xylose, and thusthe presence of a regulatory machinery to recognize the xylA promoter inthe other Streptococcus species is not likely. Therefore we constructedboth xylose induction systems but only tested the inducibility ofmCherry with the P_(Idha+XylA) system.

In order to determine mCherry inducible expression by xylose, mid-logcultures of cells with the plasmid (pBAV1KT5-XylR-mCherry-P_(Idha+XylA))were induced with different concentrations of xylose. Six hours afterthe induction we measured mCherry fluorescence in the cultures, where weobserved substantially higher overall expression levels in cellscarrying the plasmid (FIG. 6). It is worth noticing that the systemshowed a substantial level of basal expression even in the cultureswhere xylose was not added. This means that the system is ‘leaky’ and incontext of the kill-array this can lead to cell death even before thesystem is induced with xylose. However, in the subsequent course of thisstudy we used both versions of the plasmid(pBAV1KT5-XylR-mCherry-P_(Idha+XylA) andpBAV1KT5-XylR-mCherry-P_(xylA)).

2.3 Design of CRISPR/CAS9 Array

In order to determine if the genomic targeting spacers in a CRISPR arraycan cause death in S. thermophilus LMD-9, we inserted the CRISPR arraywe designed into the two xylose inducible systems previously constructed(pBAV1KT5-XylR-mCherry-P_(Idha+XylA) andpBAV1KT5-XylR-mCherry-P_(XylA)). In these plasmids we replaced mCherrywith the gBlock containing the CRISPR array (FIG. 7). The variant withthe P_(Idha+XylA) promoter was expected to be stronger and have a higherbasal activity than the P_(xylA) (Xie et al. 2013).

2.4 Inhibition of Bacterial Population Growth Using Endogenous Cas9

After we constructed the plasmids in E. coli, we transformed theplasmids into S. thermophilus. This would allow us to determine if wecould cause cell death of a specific bacterial species. Interestingly,bacterial host population size (indicated by growing bacteria andcounting colony numbers on agar plates) in S. thermophilus exposed tothe plasmid containing the strong P_(Idh+XylA) hybrid promoter was10-fold less when compared to S. thermophilus exposed to the plasmidcontaining the weak, normal P_(xylA) promoter (FIG. 8; 52 colonies withthe strong array expression versus 556 colonies with weak arrayexpression, 10.7-fold difference), the 2 strains having been transformedin parallel using the same batch of electrocompetent S. thermophiluscells. This suggests to us that the plasmid carrying the CRISPR arraytargeting S. thermophilus genes is able to kill the cells using theendogenous Cas nuclease and RNase III, thereby inhibiting populationgrowth by 10-fold.

We expect that weak array expression in host cells transformed by theplasmid comprising the P_(xylA) promoter led to a degree of cellkilling, albeit much less than with the strong promoter plasmid. Weexpect that population growth inhibition that is greater than theobserved 10-fold inhibition would be determined if a comparison of theactivity of strong array expression was made with S thermophilus that isnot exposed to any array-encoding plasmid (such as bacteria directlyisolated from gut microbiota). Thus, we believe that array (or singleguide RNA) expression in host cells for harnessing endogenous Casnuclease will be useful for providing effective growth inhibition oftarget host cells in environmental, medical and other settings mentionedherein. Co-administration of antibiotic may also be useful to enhancethe growth inhibition, particularly when one or more antibioticresistance genes are targeted.

3. Discussion and Outlook

In this study we set out to design a CRISPR-array to specifically killS. thermophilus using the endogenous Cas9 system. In order to gaincontrol over the killing signal we sought to apply an inducible systemthat can be applied in S. thermophilus. The xylose inducible XylR systemfrom B. megaterium was previously applied in S. mutans (Xie, 2013) butnot in S. thermophilus. In this study we demonstrated the functionalityof the xylR induction system using the designed XylR-mCherry-Pldhacircuit in S. thermophilus. We found 0.1% wt/vol is sufficient to fullyinduce the XylR system in S. thermophilus (FIG. 6).

In order to observe abundance when co-culturing S. thermophilus and E.coli we established that supplementation of the culture media with 3 g1⁻¹ of PEA, allows for the selective growth of S. thermophilus whilelimiting the growth of E. coli (FIG. 4).

A ST1-CRISPR array, targeting the DNA polymerase III subunit alpha and atetA like gene in the S. thermophilus LMD-9 genome, was placed under thexylose inducible promoter (Xie et al. 2013). Targeting these regionsshould lead to a double strand break and thus limit S. thermophilusviability (FIG. 9). Since the engineered array was designed to target S.thermophilus genome using the endogenous CRISPR/Cas machinery to processthe encoded CRISPR array, the array is expected to have no influence ongrowth of unrelated strains such as E. coli, even similar targets couldbe found on its genome. This was successfully tested in a mixedbacterial population (simulating aspects of a human microbiota) asdiscussed in Example 3.

The demonstration of the ability to inhibit host cell growth on asurface is important and desirable in embodiments where the invention isfor treating or preventing diseases or conditions mediated or caused bymicrobiota as disclosed herein in a human or animal subject. Suchmicrobiota are typically in contact with tissue of the subject (eg, guttissue) and thus we believe that the demonstration of activity toinhibit growth of a microbiota bacterial species (exemplified byStreptococcus) on a surface supports this utility.

Example 2: Specific Microbiota Bacterial Population Growth Inhibition inDifferent Strains

Example 1 demonstrated specific growth inhibition of Streptococcusthermophilus LMD-9. Here we demonstrate growth inhibition can also beobtained in a second strain: Streptococcus thermophilus DSM 20617.Methods described in Example 1 were, therefore, applied to the latterstrain (except that selective media for S. thermophilus DSM 20617 was THmedia supplemented with 2.5 g 1⁻¹ of 2-phenylethanol (PEA)).

Streptococcus thermophilus DSM 20617 transformed with the CRISPR arrayplasmids were incubated for recovery in liquid media for a period of 3hours at 37° C. that would allow for expression of kanamycin resistance.After a recovery period, cells were plated in different selection mediain presence of 1% xylose in order to induce cell death, and withoutxylose as a control (FIG. 10). It is evident that; (1) by xyloseinduction the growth of S. thermophilus can be inhibited (around 10-foldfor the ‘strong’ promoter plasmid versus control), (2) the ‘strong’system (pBAV1KT5-XylR-CRISPR-P_(IdhA)) results in more growth reductionthan the ‘weak’ system (pBAV1KT5-XylR-CRISPR-P_(xylA))

Example 3: Selective Bacterial Population Growth Inhibition in a MixedConsortium of Different Microbiota Species

We next demonstrated selective growth inhibition of a specific bacterialspecies in a mixed population of three species. We selected speciesfound in gut microbiota of humans and animals (S thermophilus DSM20617(T), Lactobacillus lactis and E coli). We included twogram-positive species (the S thermophilus and L lactis) to see if thiswould affect the ability for selective killing of the former species;furthermore to increase difficulty (and to more closely simulatesituations in microbiota) L lactis was chosen as this is aphylogenetically-related species to S thermophilus (as indicated by high16s ribosomal RNA sequence identity between the two species). The Sthermophilus and L lactis are both Firmicutes. Furthermore, to simulatemicrobiota, a human commensal gut species (E coli) was included.

1. Materials & Methods

Methods as set out in Example 1 were used (except that selective mediawas TH media supplemented with 2.5 g 1¹ of 2-phenylethanol (PEA)).

1.1 Preparation of Electro-Competent L. lactis Cells

Overnight cultures of L. lactis in TH media supplemented with 0.5 Msucrose and 1% glycine were diluted 100-fold in 5 ml of the same mediaand grown at 30° C. to an OD₆₀₀ between 0.2-0.7 (approximately 2 hoursafter inoculation). The cells were collected at 7000×g for 5 min at 4°C. and washed three times with 5 ml of ice cold wash buffer (0.5 Msucrose+10% glycerol). After the cells were washed, they were suspendedto an OD₆₀₀ of 15-30 in electroporation buffer (0.5 M sucrose, 10%glycerol and 1 mM MgCl₂). The cells in the electroporation buffer werekept at 4° C. until use (within one hour) or aliquot 50 μl in eppendorftubes, freezing them in liquid nitrogen and stored at −80° C. for lateruse.

Electroporation conditions for all species were as described in Example1.

1.2 Activation of CRISPR Array: Consortium Experiments.

S. thermophilus DSM 20617, L. lactis MG1363 and E. coli TOP10 weregenetically transformed with the plasmid containing the CRISPR arraytargeting the DNA polymerase III and tetA of S. thermophilus. Aftertransformation all cells were grown alone and in co-culture for 3 hoursat 37° C. allowing for recovery to develop the antibiotic resistanceencoded in the plasmid. We decided to use transformation efficiency as aread out of CRISPR-encoded growth inhibition. Therefore, after allowingthe cells for recovery the cultures were plated in TH media, THsupplemented with PEA and MacConkey agar all supplemented withKanamycin, and induced by 1% xylose.

2. Results

2.0 Phylogenetic Distance Between L. lactis, E. Coli and S. thermophilus

The calculated sequence similarity in the 16S rRNA-encoding DNA sequenceof the S. thermophilus and L. lactis was determined as 83.3%. Thefollowing 16S sequences were used: E. coli: AB030918.1, S. thermophilus:AY188354.1, L. lactis: AB030918. The sequences were aligned with needle(http://www.ebi.ac.uk/Tools/psa/emboss_needle/nucleotide.html) with thefollowing parameters: -gapopen 10.0-gapextend 0.5-endopen 10.0-endextend0.5-aformat3 pair—snucleotide1-snucleotide2. FIG. 11 shows themaximum-likelihood phylogenetic tree of 16S sequences from S.thermophilus, L. lactis and E. coli.

2.1 Growth Condition and Selective Media

S. thermophilus and L. lactis are commonly used in combination in manyfermented foods and yoghurt. We chose these strains since they arecommonly known to be gut microbes that form an intimate association withthe host and previous characterizations of the 16S ribosomal RNA regionof S. thermophilus and L. lactis have shown that these organisms arephylogenetically closely related (Ludwig et al., 1995). In parallel wealso evaluated the growth of E. coli for our mixed population co-cultureexperiments, since this organism is also commonly found in gut microbecommunities. We first set out to establish the bacterial strains andcultivation protocol that would support growth for all strains weplanned to use for the co-cultivation experiments. We found that allstrains were able to support growth in TH broth at 37° C. (FIG. 3).

Distinguishing the different bacteria from a mixed culture is importantin order to determine cell number of the different species. WithMacConkey agar is possible to selectively grow E. coli, however there isno specific media for selective growth of S. thermophilus. PEA agar is aselective medium that is used for the isolation of gram-positive (S.thermophilus) from gram-negative (E. coli). Additionally, differentconcentrations of PEA partially inhibit the growth of the differentgrams positive species and strains, which allow for selection betweenthe other gram-positive bacteria used in this work. Using 2.5 g 1¹ ofPEA proved to selectively grow S. thermophilus while limiting growth ofL. lactis and E. coli.

All strains were transformed with a plasmid that used the vectorbackbone of pBAV1KT5 that has a kanamycin selection marker; we foundthat using media supplemented with 30 ug ml⁻¹ of kanamycin was enough togrow the cells while keeping the plasmid.

2.3 Transformation & Selective Growth Inhibition in a Mixed Population

We transformed S. thermophilus, L. lactis and E. coli with plasmidcontaining the CRISPR array and cultured them in a consortium of all thebacterial species combined in equal parts, which would allow us todetermine if we could cause cell death specifically in S. thermophilus.We transformed all the species with either thepBAV1KT5-XylR—CRISPR-P_(XylA) or pBAV1KT5-XylR-CRISPR-P_(Idha+XylA)plasmid.

‘FIG. 12 shows the selective S thermophilus growth inhibition in aco-culture of E. coli, L. lactis and S. thermophilus harboring eitherthe pBAV1KT5-XylR-CRISPR-P_(xylA) or the pBAV1KT5-XylR—CRISPR-P_(IdhA+XylA) plasmid. No growth difference is observedbetween E. coli harboring the pBAV1KT5-XylR-CRISPR-P_(xylA) or thepBAV1KT5-XylR-CRISPR-P_(IdhA+XylA) plasmid (middle column). However, S.thermophilus (selectively grown on TH agar supplemented with 2.5 gl⁻¹PEA, last column) shows a decrease in transformation efficiency betweenthe pBAV1KT5-XylR—CRISPR-P_(xylA) (strong) or thepBAV1KT5-XylR-CRISPR-P_(IdhA+XylA) (weak) plasmid as we expected. Wethus demonstrated a selective growth inhibition of the target Sthermophilus sub-population in the mixed population of cells.

REFERENCES

-   1. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Patrick    Boyaval, Moineau, S., . . . Horvath, P. (2007). CRISPRProvides    Acquired Resistance Against Viruses in Prokaryotes. Science,    315(March), 1709-1712.-   2. Bryksin, A. V, & Matsumura, I. (2010). Rational design of a    plasmid origin that replicates efficiently in both gram-positive and    gram-negative bacteria. PloS One, 5(10), e13244.-   3. Chan C T Y, Lee J W, Cameron D E, Bashor C J, Collins J J:    “Deadman” and “Passcode” microbial kill switches for bacterial    containment. Nat Chem Biol 2015, 12(Dec.):1-7.-   4. Horvath, P., Romero, D. A., Coûté-Monvoisin, A.-C., Richards, M.,    Deveau, H., Moineau, S., . . . Barrangou, R. (2008). Diversity,    activity, and evolution of CRISPR loci in Streptococcus    thermophilus. Journal of Bacteriology, 190(4), 1401-12.-   5. Ludwig, E. S., Klipper, R., Magrum L., Wose C., &    Stackebrandt, E. (1985). The phylogenetic position of Streptococcus    and Enterococcus. Journal of Gencwl Microhiologj., 131, 543-55 1.-   6. Mercenier, A. (1990). Molecular genetics of Streptococcus    thermophilus. FEMS Microbiology Letters, 87(1-2), 61-77. \-   7. Samaržija, D., Antunac, N., & Havranek, J. (2001). Taxonomy,    physiology and growth of Lactococcus lactis: a review. Mljekarstvo,    51(1), 35-48. Retrieved from-   8. Sapranauskas, R., Gasiunas, G., Fremaux, C., Barrangou, R.,    Horvath, P., & Siksnys, V. (2011). The Streptococcus thermophilus    CRISPR/Cas system provides immunity in Escherichia coli. Nucleic    Acids Research, 39(21), 9275-9282.-   9. Somkuti, G. A., & Steinberg, D. H. (1988). Genetic transformation    of Streptococcus thermophilus by electroporation. Biochimie, 70(4),    579-585-   10. Sorg, R. A., Kuipers, O. P., & Veening, J.-W. (2014). Gene    expression platform for synthetic biology in the human pathogen    Streptococcus pneumoniae. ACS Synthetic Biology, 4(3), 228-239.-   11. Suvorov, a. (1988). Transformation of group A streptococci by    electroporation. FEMS Microbiology Letters, 56(1), 95-99.-   12. Xie, Z., Qi, F., & Merritt, J. (2013). Development of a tunable    wide-range gene induction system useful for the study of    streptococcal toxin-antitoxin systems. Applied and Environmental    Microbiology, 79(20), 6375-84.-   13. Zhang, X. Z., & Zhang, Y. H. P. (2011). Simple, fast and    high-efficiency transformation system for directed evolution of    cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1),    98-105.

Example 4: Altering the Ratio of Clostridium dificile in a Mixed GutMicrobiota Population

Alteration of the ratio of bacteria will be performed according to thepresent example, which is described by reference to knocking-downClostridium dificile bacteria in a mixed gut microbiota sample. Thesample will contain Bacteroides and metronidazole (MTZ)-resistant Cdificile strain 630 sub-populations. Ex vivo the mixed population iscombined with a population of carrier bacteria (Lactobacillusacidophilus La-14 and/or La-5) that have been engineered to containCRISPR arrays.

Each CRISPR array is comprised on a plasmid that is compatible with thecarrier bacterium and C dificile cells. The array is comprised by aBacteroides thetaiotamicron CTnDot transposon that also comprises oriT,an intDOT sequence, a tetQ-rteA-rteB operon, rteC and the operonxis2c-xis2d-orf3-exc. In one experiment, mob and tra operons areexcluded (instead relying on these supplied by Bacteroides cells towhich the transposons are transferred in the mixture combined with thecarrier bacteria). In another experiment, the mob and tra operons areincluded in the transposons.

Protein translocation across the cytoplasmic membrane is an essentialprocess in all bacteria. The Sec system, comprising at its core anATPase, SecA, and a membrane channel, SecYEG, is responsible for themajority of this protein_transport. A second parallel Sec system hasbeen described in a number of Gram-positive species. This accessory Secsystem is characterized by the presence of a second copy of theenergizing ATPase, SecA2; where it has been studied, SecA2 isresponsible for the translocation of a subset of Sec substrates. Incommon with many pathogenic Gram-positive species, Clostridium difficilepossesses two copies of SecA. Export of the S-layer proteins (SLPs) andan additional cell wall protein (CwpV) is dependent on SecA2.Accumulation of the cytoplasmic precursor of the SLPs SlpA and othercell wall proteins is observed in cells expressing dominant-negativesecA1 or secA2 alleles, concomitant with a decrease in the levels ofmature SLPs in the cell wall. Furthermore, expression of eitherdominant-negative allele or antisense RNA knockdown of SecA1 or SecA2dramatically impairs growth, indicating that both Sec systems areessential in C. difficile.

C. difficile Strain 630 (epidemic type X) has a single circularchromosome with 4,290,252 bp (G+C content=29.06%) and a circular plasmidwith 7,881 bp (G+C content=27.9%). The whole genome has been sequencedand found that 11% of the genome consists of mobile genetic elementssuch as conjugative transposons. These elements provide C. difficilewith the genes responsible for its antimicrobial resistance, virulence,host interaction and the production of surface structures. For example,the cdeA gene of C. difficile produces a multidrug efflux pump which wasshown to be homologous to known efflux transporters in the multidrug andtoxic compound extrusion (MATE) family. The protein facilitatesenergy-dependent and sodium-coupled efflux of drugs from cells. Inaddition, the cme gene in C. difficile has been shown to providemultidrug resistance in other bacteria.

The array comprises a R1-S1-R1′ CRISPR unit (spacer flanked by twoCRISPR repeats) for targeting a sequence in an essential gene (SecA2) ofC dificile cells. In another experiment, targeting is to the cdeA genein the presence of MTZ and optionally one or more other anti-C dificileantibiotics. Each spacer (S) comprises a 20mer nucleotide sequence ofthe SecA or cdeA gene, wherein the sequence comprises a PAM of a Cdificile strain 630 CRISPR/Cas system that is cognate to the repeatsequences. Each repeat is identical to a C dificile strain 630 repeat.

The repeats function with Cas that is endogenous to the C dificile cellsin the mixed population. The mixed population of bacteria is retrievedas an ex vivo sample from a stool sample of a human patient sufferingfrom C dificile infection. The mixed population is mixed with thecarrier bacteria in vitro and incubated at 37 degrees centigrade underanaerobic conditions to simulate gut conditions in the presence oftetracycline. It is expected that transposons containing the CRISPRarrays will be transferred to Bacteroides and C dificile cells in themixture. Furthermore, it is expected that the target sites in the lattercells will be cut by Cas nuclease action, thus reducing the proportionof C dificile in the mixed population (and increasing the ratio ofBacteroides versus C dificile).

In a follow-on experiment, a drink is produced comprising the carrierbacteria and this is consumed by the human patient once or twice forseveral consecutive days with or without an ant-acid. The patient isalso administered with tetracycline during the treatment period. It isexpected that stool analysis will reveal that the proportion of Cdificile in the stool samples will reduce (and the ratio of Bacteroidesversus C dificile will increase).

Example 5: Vector-Encoded System for Selective Species & Strain GrowthInhibition in a Mixed Bacterial Consortium

In Example 3 we surprisingly established the possibility of harnessingendogenous Cas nuclease activity in host bacteria for selectivepopulation growth inhibition in a mixed consortium of different species.We next explored the possibility of instead using vector-encoded Casactivity for selective population growth inhibition in a mixedconsortium of different species. We demonstrated selective growthinhibition of a specific bacterial species in a mixed population ofthree different species, and further including a strain alternative tothe target bacteria. We could surprisingly show selective growthinhibition of just the target strain of the predetermined targetspecies. Furthermore, the alternative strain was not targeted by thevector-encoded CRISPR/Cas system, which was desirable for establishingthe fine specificity of such vector-borne systems in a mixed bacterialconsortium that mimicked human or animal gut microbiota elements.

We selected species found in gut microbiota of humans and animals(Bacillus subtilis, Lactobacillus lactis and E coli). We included twostrains of the human commensal gut species, E coli. We thought it ofinterest to see if we could distinguish between closely related strainsthat nevertheless had sequence differences that we could use to targetkilling in one strain, but not the other. This was of interest as somestrains of E coli in microbiota are desirable, whereas others may beundesirable (eg, pathogenic to humans or animals) and thus could betargets for Cas modification to knock-down that strain.

1. Material and Methods

1.1. Plasmids and Strains

See Tables 7 and 8. All strains were cultivated in Todd-Hewitt broth(TH) (T1438 Sigma-Aldrich), in aerobic conditions and at 37° C., unlesselsewhere indicated. The strains were stored in 25% glycerol at −80° C.

The self-targeting sgRNA-Cas9 complex was tightly regulated by atheophylline riboswitch and the AraC/P_(BAD) expression systemrespectively. Tight regulation of Cas9 is desired in order to be carriedstably in E. coli. The plasmid contained the exogenous Cas9 fromStreptococcus pyogenes with a single guide RNA (sgRNA) targeting E.coli's K-12 strains. Therefore K-12 derived strains TOP10 wassusceptible to double strand self-cleavage and consequent death when thesystem was activated. E. coli strains like Nissle don't have the sametarget sequence therefore they were unaffected by the sgRNA-Cas9activity. See Tables 9-11 below, which show sequences used in Example 9.We chose a target sequence (ribosomal RNA-encoding sequence) that isconserved in the target cells and present in multiple copies (7 copies),which increased the chances of cutting host cell genomes in multipleplaces to promote killing using a single gRNA design.

FIG. 13 shows regulators controlling the expression of spCas9 and theself-targeting sgRNA targeting the ribosomal RNA subunit 16s.

1.2. Differential Growth Media

All strains were grown on TH media at 37° C. for 20 hours. Selectivemedia for B. subtilis was TH media supplemented with 2.5 g 1⁻¹ of2-phenylethanol (PEA). PEA was added to the media and autoclaved at 121°C. for 15 minutes at 15 psi. Agar plates were prepared by adding 1.5%(wt/vol) agar to the corresponding media.

1.3. Cloning

E. coli (ONE SHOT® ThermoFischer TOP10 Chemically Competent cells) wasused in all subcloning procedures. PCR was carried out using PHUSION™polymerase. All PCR products were purified with NUCLEOSPIN™ Gel and PCRClean-up by MACHEREY-NAGEL™ following the manufacturer's protocol. Thepurified fragments were digested with restriction enzyme DpnI in 1× FDbuffer with 1 μl enzyme in a total volume of 34 μl. The digestedreaction was again purified with Nucleospin Gel and PCR Clean-up byMacherey-Nagel following the manufacturer's protocol. Gibson assemblywas performed in 10 μl reactions following the manufacturer's protocol(NewEngland Biolab).

Plasmid DNA was prepared using Qiagen kits according to themanufacturer's instructions. Modifications for Gram-positive strainsincluded growing bacteria in a medium supplemented with 0.5% glycine andlysozyme to facilitate cell lysis.

-   1.4. Transformation-   1.4.1 Electro-Competent E. coli Cells and Transformation

Commercially electrocompetent cells were used for cloning and theexperiments (ONE SHOT® ThermoFischer TOP10 electrompetent E. coli).Electroporation was done using standard settings: 1800 V, 25 μF and 200Ωusing an Electro Cell Manipulator (BTX Harvard Apparatus ECM630).Following the pulse, 1 ml LB-SOC media was added and the cells wereincubated at 37° C. for 1 hour. The transformed cells were plated inLB-agar containing the corresponding antibiotics.

1.5. Activation of sgRNA-Cas9 in E. coli and Consortium Experiments.

E. coli TOP10 and Nissle both with the plasmid containing the sgRNAtargeting the ribosomal RNA-encoding sequence of K-12 derived strainsand the other bacteria were grown overnight in 3 ml of TH broth. Thenext day the cells were diluted to ˜OD 0.5 and next 10-fold seriallydiluted in TH media and using a 96-well replicator (METTLER TOLEDOLIQUIDATOR™ 96) 4 μL volume drops were spotted on TH agar, TH agar withinducers (1% arabinose and 2 mM theophylline), TH agar supplemented with2.5 g 1⁻¹ PEA and MacConkey agar supplemented with 1% maltose. Theplates were incubated for 20 h at 37° C. and the colony forming units(CFU) were calculated from triplicate measurements.

2. Results

2.1 Specific Targeting of E. coli Strains Using an Exogenous CRISPR-Cas9System

We first tested if the system could differentiate between two E. colistrains by introducing the killing system in both E. coli TOP10 andNissle.

2.1 Targeting of E. coli Using an Exogenous CRISPR-Cas9 System in aMixed Culture

Serial dilutions of overnight cultures were done in duplicate for bothE. coli strains, B. subtilis, L. lactis, and in triplicate for the mixedcultures. All strains were grown at 37° C. for 20 hours in selectiveplates with and without the inducers. Induction of the system activatesthe sgRNA-Cas9 targeting K-12 derived strains, while leaving intact theother bacteria.

Distinguishing the different bacteria from a mixed culture is importantin order to determine cell numbers of the different species anddetermine the specific removal of a species. MacConkey agar selectivelygrows E. coli, PEA agar is a selective medium that is used for theisolation of gram-positive (B. subtilis) from gram-negative (E. coli).Additionally, we found that different concentrations of PEA partiallyinhibit the growth of other gram positives. 2.5 g 1¹ of PEA proved toselectively grow B. subtilis while limiting growth of E. coli and L.lactis.

FIG. 14 shows specific targeting of E. coli strain by the inducibe,exogenous, vector-borne CRISPR-Cas system. The sgRNA target the genomeof K-12 derived E. coli strain E. coli TOP10, while the other E. colistrain tested was unaffected.

FIG. 15 shows spot assay with serial dilutions of individual bacterialspecies used in this study and mixed culture in TH agar withoutinduction of the CRISPR-Cas9 system.

FIG. 16 shows a spot assay of the dilution 10³ on different selectivemedia. TH with 2.5 g 1⁻¹ PEA is a selective media for B. subtilis alone.MacConkey supplemented with maltose is a selective and differentialculture medium for bacteria designed to selectively isolateGram-negative and enteric bacilli and differentiate them based onmaltose fermentation. Therefore TOP10 ΔmalK mutant makes white colonieson the plates while Nissle makes pink colonies; A is E coli ΔmalK, B isE coli Nissile, C is B subtilis, D is L lactis, E is mixed culture; theimages at MacConkey−/B and E appear pink; the images at MacConkey+/B andE appear pink. FIG. 17 shows selective growth of the bacteria used inthis study on different media and selective plates. It can be seen thatwe clearly, selectively killed the target E coli strain (“E coli” onx-axis in FIG. 17) in the mixed population, whereas the other relatedstrain (“E coli—Nissle”) was not similarly killed Killing of the targetstrain in the mixed population was 1000-fold in this experiment.

REFERENCES

-   1. Zhang, X. Z., & Zhang, Y. H. P. (2011). Simple, fast and    high-efficiency transformation system for directed evolution of    cellulase in Bacillus subtilis. Microbial Biotechnology, 4(1),    98-105. http://doi.org/10.1111/j.1751-7915.2010.00230.x-   2. Wegmann, U., O'Connell-Motherway, M., Zomer, A., Buist, G.,    Shearman, C., Canchaya, C., . . . Kok, J. (2007). Complete genome    sequence of the prototype lactic acid bacterium Lactococcus lactis    subsp.cremoris MG1363. Journal of Bacteriology, 189(8), 3256-70.    http://doi.org/10.1128/JB.01768-06

TABLE 1 EXAMPLE BACTERIA Abiotrophia Acidocella ActinomycesAlkalilimnicola Aquaspirillum Abiotrophia Acidocella Actinomyces bovisAlkalilimnicola Aquaspirillum defectiva aminolytica Actinomycesehrlichii polymorphum Acaricomes Acidocella facilis denticolensAlkaliphilus Aquaspirillum Acaricomes Acidomonas ActinomycesAlkaliphilus putridiconchylium phytoseiuli Acidomonas europaeusoremlandii Aquaspirillum Acetitomaculum methanolica ActinomycesAlkaliphilus serpens Acetitomaculum Acidothermus georgiae transvaalensisAquimarina ruminis Acidothermus Actinomyces Allochromatium AquimarinaAcetivibrio cellulolyticus gerencseriae Allochromatium laterculaAcetivibrio Acidovorax Actinomyces vinosum Arcanobacteriumcellulolyticus Acidovorax hordeovulneris Alloiococcus ArcanobacteriumAcetivibrio anthurii Actinomyces Alloiococcus otitis haemolyticumethanolgignens Acidovorax caeni howellii Allokutzneria ArcanobacteriumAcetivibrio Acidovorax Actinomyces Allokutzneria albata pyogenesmultivorans cattleyae hyovaginalis Altererythrobacter ArchangiumAcetoanaerobium Acidovorax citrulli Actinomyces AltererythrobacterArchangium Acetoanaerobium Acidovorax israelii ishigakiensis gephyranoterae defluvii Actinomyces Altermonas Arcobacter AcetobacterAcidovorax johnsonii Altermonas Arcobacter butzleri Acetobacter acetidelafieldii Actinomyces haloplanktis Arcobacter Acetobacter Acidovoraxfacilis meyeri Altermonas cryaerophilus cerevisiae AcidovoraxActinomyces macleodii Arcobacter Acetobacter konjaci naeslundiiAlysiella halophilus cibinongensis Acidovorax Actinomyces neuiiAlysiella crassa Arcobacter Acetobacter temperans Actinomyces Alysiellafiliformis nitrofigilis estunensis Acidovorax odontolyticus AminobacterArcobacter Acetobacter valerianellae Actinomyces oris Aminobacterskirrowii fabarum Acinetobacter Actinomyces aganoensis ArhodomonasAcetobacter Acinetobacter radingae Aminobacter Arhodomonas ghanensisbaumannii Actinomyces aminovorans aquaeolei Acetobacter Acinetobacterslackii Aminobacter Arsenophonus indonesiensis baylyi Actinomycesniigataensis Arsenophonus Acetobacter Acinetobacter turicensisAminobacterium nasoniae lovaniensis bouvetii Actinomyces AminobacteriumArthrobacter Acetobacter Acinetobacter viscosus mobile Arthrobacteragilis malorum calcoaceticus Actinoplanes Aminomonas Arthrobacter albusAcetobacter Acinetobacter Actinoplanes Aminomonas Arthrobacternitrogenifigens gerneri auranticolor paucivorans aurescens Acetobacteroeni Acinetobacter Actinoplanes Ammoniphilus Arthrobacter Acetobacterhaemolyticus brasiliensis Ammoniphilus chlorophenolicus orientalisAcinetobacter Actinoplanes oxalaticus Arthrobacter Acetobacter johnsoniiconsettensis Ammoniphilus citreus orleanensis Acinetobacter juniiActinoplanes oxalivorans Arthrobacter Acetobacter Acinetobacterdeccanensis Amphibacillus crystallopoietes pasteurianus lwoffiActinoplanes Amphibacillus Arthrobacter Acetobacter Acinetobacterderwentensis xylanus cumminsii pornorurn parvus Actinoplanes AmphriteaArthrobacter Acetobacter Acinetobacter digitatis Amphritea balenaeglobiformis senegalensis radioresistens Actinoplanes Amphritea japonicaArthrobacter Acetobacter Acinetobacter durhamensis Amycolatopsishistidinolovorans xylinus schindleri Actinoplanes Amycolatopsis albaArthrobacter ilicis Acetobacterium Acinetobacter soli ferrugineusAmycolatopsis Arthrobacter luteus Acetobacterium AcinetobacterActinoplanes albidoflavus Arthrobacter bakii tandoii globisporusAmycolatopsis methylotrophus Acetobacterium Acinetobacter Actinoplanesazurea Arthrobacter carbinolicum tjernbergiae humidus Amycolatopsismysorens Acetobacterium Acinetobacter Actinoplanes coloradensisArthrobacter dehalogenans towneri italicus Amycolatopsis nicotianaeAcetobacterium Acinetobacter Actinoplanes lurida Arthrobacter fimetariumursingii liguriensis Amycolatopsis nicotinovorans AcetobacteriumAcinetobacter Actinoplanes mediterranei Arthrobacter malicum venetianuslobatus Amycolatopsis oxydans Acetobacterium Acrocarpospora Actinoplanesrifamycinica Arthrobacter paludosum Acrocarpospora missouriensisAmycolatopsis pascens Acetobacterium corrugata Actinoplanes rubidaArthrobacter tundrae Acrocarpospora palleronii Amycolatopsisphenanthrenivorans Acetobacterium macrocephala Actinoplanes sulphureaArthrobacter wieringae Acrocarpospora philippinensis Amycolatopsispolychromogenes Acetobacterium pleiomorpha Actinoplanes tolypomycinaAtrhrobacter woodii Actibacter rectilineatus Anabaena protophormiaeAcetofilamentum Actibacter Actinoplanes Anabaena cylindrica ArthrobacterAcetofilamentum sediminis regularis Anabaena flosaquaepsychrolactophilus rigidum Actinoalloteichus Actinoplanes Anabaenavariabilis Arthrobacter Acetohalobium Actinoalloteichus teichomyceticusAnaeroarcus ramosus Acetohalobium cyanogriseus Actinoplanes AnaeroarcusArthrobacter arabaticum Actinoalloteichus utahensis burkinensissulfonivorans Acetomicrobium hymeniacidonis ActinopolysporaAnaerobaculum Arthrobacter Acetomicrobium ActinoalloteichusActinopolyspora Anaerobaculum sulfureus faecale spitiensis halophilamobile Arthrobacter Acetomicrobium Actinobaccillus ActinopolysporaAnaerobiospirillum uratoxydans flavidum Actinobacillus mortivallisAnaerobiospirillum Arthrobacter Acetonema capsulatus Actinosynnemasucciniciproducens ureafaciens Acetonema longum ActinobacillusActinosynnema Anaerobiospirillum Arthrobacter Acetothermus delphinicolamirum thomasii viscosus Acetothermus Actinobacillus ActinotaleaAnaerococcus Arthrobacter paucivorans hominis Actinotalea Anaerococcuswoluwensis Acholeplasma Actinobacillus fermentans hydrogenalis AsaiaAcholeplasma indolicus Aerococcus Anaerococcus Asaia bogorensis axanthumActinobacillus Aerococcus lactolyticus Asanoa Acholeplasma lignieresiisanguinicola Anaerococcus Asanoa ferruginea brassicae ActinobacillusAerococcus urinae prevotii Asticcacaulis Acholeplasma minor AerococcusAnaerococcus Asticcacaulis cavigenitalium Actinobacillus urinaeequitetradius biprosthecium Acholeplasma muris Aerococcus AnaerococcusAsticcacaulis equifetale Actinobacillus urinaehominis vaginalisexcentricus Acholeplasma pleuropneumoniae Aerococcus AnaerofustisAtopobacter granularum Actinobacillus viridans Anaerofustis AtopobacterAcholeplasma porcinus Aeromicrobium stercorihominis phocae hippikonActinobacillus Aeromicrobium Anaeromusa Atopobium Acholeplasma rossiierythreum Anaeromusa Atopobium fossor laidlawii Actinobacillus Aeromonasacidaminophila Atopobium Acholeplasma scotiae Aeromonas Anaeromyxobacterminutum modicum Actinobacillus allosaccharophila AnaeromyxobacterAtopobium Acholeplasma seminis Aeromonas dehalogenans parvulum morumActinobacillus bestiarum Anaerorhabdus Atopobium rimae Acholeplasmasuccinogenes Aeromonas caviae Anaerorhabdus Atopobium vaginaemultilocale Actinobaccillus Aeromonas furcosa AureobacteriumAcholeplasma suis encheleia Anaerosinus Aureobacterium oculiActinobacillus Aeromonas Anaerosinus barkeri Acholeplasma ureaeenteropelogenes glycerini Aurobacterium palmae Actinobaculum AeromonasAnaerovirgula Aurobacterium Acholeplasma Actinobaculum eucrenophilaAnaerovirgula liquefaciens parvum massiliense Aeromonas multivoransAvibacterium Acholeplasma Actinobaculum ichthiosmia AncalomicrobiumAvibacterium avium pleciae schaalii Aeromonas AncalomicrobiumAvibacterium Acholeplasma Actinobaculum jandaei adetum gallinarum vitulisuis Aeromonas media Ancylobacter Avibacterium Achromobacter ActinomycesAeromonas Ancylobacter paragallinarum Achromobacter urinale popoffiiaquaticus Avibacterium denitrificans Actinocatenispora Aeromonas sobriaAneurinibacillus volantium Achromobacter Actinocatenispora Aeromonasveronii Aneurinibacillus Azoarcus insolitus rupis Agrobacteriumaneurinilyticus Azoarcus indigens Achromobacter ActinocatenisporaAgrobacterium Aneurinibacillus Azoarcus piechaudii thailandicagelatinovorum migulanus tolulyticus Achromobacter ActinocatenisporaAgrococcus Aneurinibacillus Azoarcus ruhlandii sera Agrococcusthermoaerophilus toluvorans Achromobacter Actinocorallia citreusAngiococcus Azohydromonas spanius Actinocorallia Agrococcus AngiococcusAzohydromonas Acidaminobacter aurantiaca jenensis disciformis australicaAcidaminobacter Actinocorallia Agromonas Angulomicrobium Azohydromonashydrogenoformans aurea Agromonas Angulomicrobium lata AcidaminococcusActinocorallia oligotrophica tetraedrale Azomonas Acidaminococcuscavernae Agromyces Anoxybacillus Azomonas agilis fermentansActinocorallia Agromyces Anoxybacillus Azomonas insignis Acidaminococcusglomerata fucosus pushchinoensis Azomonas intestini ActinocoralliaAgromyces Aquabacterium macrocytogenes Acidicaldus herbida hippuratusAquabacterium Azorhizobium Acidicaldus Actinocorallia Agromyces communeAzorhizobium organivorans libanotica luteolus Aquabacterium caulinodansAcidimicrobium Actinocorallia Agromyces parvum AzorhizophilusAcidimicrobium longicatena mediolanus Azorhizophilus ferrooxidansActinomadura Agromyces paspali Acidiphilium Actinomadura alba ramosusAzospirillum Acidiphilium Actinomadura Agromyces Azospirillumacidophilum atramentaria rhizospherae brasilense AcidiphiliumActinomadura Akkermansia Azospirillum angustum bangladeshensisAkkermansia halopraeferens Acidiphilium Actinomadura muciniphilaAzospirillum cryptum catellatispora Albidiferax irakense AcidiphiliumActinomadura Albidiferax Azotobacter multivorum chibensis ferrireducensAzotobacter Acidiphilium Actinomadura Albidovulum beijerinckiiorganovorum chokoriensis Albidovulum Azotobacter AcidiphiliumActinomadura inexpectatum chroococcum rubrum citrea AlcaligenesAzotobacter Acidisoma Actinomadura Alcaligenes nigricans Acidisomacoerulea denitrificans Azotobacter sibiricum Actinomadura Alcaligenessalinestris Acidisoma tundrae echinospora faecalis AzotobacterAcidisphaera Actinomadura Alcanivorax vinelandii Acidisphaera fibrosaAlcanivorax rubrifaciens Actinomadura borkumensis Acidithiobacillusformosensis Alcanivorax Acidithiobacillus Actinomadura jadensisalbertensis hibisca Algicola Acidithiobacillus Actinomadura Algicolacaldus kijaniata bacteriolytica Acidithiobacillus ActinomaduraAlicyclobacillus ferrooxidans latina Alicyclobacillus AcidithiobacillusActinomadura disulfidooxidans thiooxidans livida AlicyclobacillusAcidobacterium Actinomadura sendaiensis Acidobacterium luteofluorescensAlicyclobacillus capsulatum Actinomadura vulcanalis macra AlishewanellaActinomadura Alishewanella madurae fetalis Actinomadura Alkalibacillusoligospora Alkalibacillus Actinomadura haloalkaliphilus pelletieriActinomadura rubrobrunea Actinomadura rugatobispora Actinomadura umbrinaActinomadura verrucosospora Actinomadura vinacea Actinomaduraviridilutea Actinomadura viridis Actinomadura yumaensis BacillusBacteroides Bibersteinia Borrelia Brevinema [see below] BacteroidesBibersteinia trehalosi Borrelia afzelii Brevinema Bacteriovorax caccaeBifidobacterium Borrelia americana andersonii Bacteriovorax BacteroidesBifidobacterium Borrelia Brevundimonas stolpii coagulans adolescentisburgdorferi Brevundimonas Bacteroides Bifidobacterium Borrelia albaeggerthii angulatum carolinensis Brevundimonas BacteroidesBifidobacterium Borrelia coriaceae aurantiaca fragilis animalis Borreliagarinii Brevundimonas Bacteroides Bifidobacterium Borrelia japonicadiminuta galacturonicus asteroides Bosea Brevundimonas BacteroidesBifidobacterium Bosea intermedia helcogenes bifidum minatitlanensisBrevundimonas Bacteroides Bifidobacterium boum Bosea thiooxidanssubvibrioides ovatus Bifidobacterium breve Brachybacterium BrevundimonasBacteroides Bifidobacterium Brachybacterium vancanneytii pectinophiluscatenulatum alimentarium Brevundimonas Bacteroides BifidobacteriumBrachybacterium variabilis pyogenes choerinum faecium BrevundimonasBacteroides Bifidobacterium Brachybacterium vesicularis salyersiaecoryneforme paraconglomeratum Brochothrix Bacteroides BifidobacteriumBrachybacterium Brochothrix stercoris cuniculi rhamnosum campestrisBacteroides suis Bifidobacterium Brachybacterium Brochothrix Bacteroidesdentium tyrofermentans thermosphacta tectus Bifidobacterium BrachyspiraBrucella Bacteroides gallicum Brachyspira Brucella canisthetaiotaomicron Bifidobacterium alvinipulli Brucella Bacteroidesgallinarum Brachyspira neotomae uniformis Bifidobacterium hyodysenteriaeBryobacter Bacteroides indicum Brachyspira Bryobacter ureolyticusBifidobacterium longum innocens aggregatus Bacteroides BifidobacteriumBrachyspira Burkholderia vulgatus magnumBifidobacterium murdochiiBurkholderia Balnearium merycicum Brachyspira ambifaria BalneariumBifidobacterium pilosicoli Burkholderia lithotrophicum minimumBradyrhizobium andropogonis Balneatrix Bifidobacterium BradyrhizobiumBurkholderia Balneatrix alpica pseudocatenulatum canariense anthinaBalneola Bifidobacterium Bradyrhizobium Burkholderia Balneola vulgarispseudolongum elkanii caledonica Barnesiella BifidobacteriumBradyrhizobium Burkholderia Barnesiella pullorum japonicum caryophylliviscericola Bifidobacterium Bradyrhizobium Burkholderia Bartonellaruminantium liaoningense cenocepacia Bartonella BifidobacteriumBrenneria Burkholderia alsatica saeculare Brenneria alni cepaciaBartonella Bifidobacterium subtile Brenneria Burkholderia bacilliformisBifidobacterium nigrifluens cocovenenans Bartonella thermophilumBrenneria quercina Burkholderia clarridgeiae Bilophila Brenneriaquercina dolosa Bartonella Bilophila wadsworthia Brenneria salicisBurkholderia doshiae Biostraticola Brevibacillus fungorum BartonellaBiostraticola tofi Brevibacillus agri Burkholderia elizabethae BizioniaBrevibacillus glathei Bartonella Bizionia argentinensis borstelensisBurkholderia grahamii Blastobacter Brevibacillus brevis glumaeBartonella Blastobacter capsulatus Brevibacillus Burkholderia henselaeBlastobacter centrosporus graminis Bartonella denitrificansBrevibacillus Burkholderia rochalimae Blastococcus choshinensiskururiensis Bartonella Blastococcus Brevibacillus Burkholderia vinsoniiaggregatus invocatus multivorans Bavariicoccus BlastococcusBrevibacillus Burkholderia Bavariicoccus saxobsidens laterosporusphenazinium seileri Blastochloris Brevibacillus BurkholderiaBdellovibrio Blastochloris parabrevis plantarii Bdellovibrio viridisBrevibacillus Burkholderia bacteriovorus Blastomonas reuszeri pyrrociniaBdellovibrio Blastomonas Brevibacterium Burkholderia exovorus natatoriaBrevibacterium silvatlantica Beggiatoa Blastopirellula abidumBurkholderia Beggiatoa alba Blastopirellula Brevibacterium stabilisBeijerinckia marina album Burkholderia Beijerinckia BlautiaBrevibacterium thailandensis derxii Blautia coccoides aurantiacumBurkholderia Beijerinckia Blautia hansenii Brevibacterium tropicafluminensis Blautia producta celere Burkholderia Beijerinckia Blautiawexlerae Brevibacterium unamae indica Bogoriella epidermidisBurkholderia Beijerinckia Bogoriella Brevibacterium vietnamiensismobilis caseilytica frigoritolerans Buttiauxella Belliella BordetellaBrevibacterium Buttiauxella Belliella baltica Bordetella aviumhalotolerans agrestis Bellilinea Bordetella Brevibacterium ButtiauxellaBellilinea bronchiseptica iodinum brennerae caldifistulae Bordetellahinzii Brevibacterium Buttiauxella Belnapia Bordetella holmesii linensferragutiae Belnapia Bordetella parapertussis BrevibacteriumButtiauxella moabensis Bordetella pertussis lyticum gaviniae BergeriellaBordetella petrii Brevibacterium Buttiauxella Bergeriella Bordetellatrematum mcbrellneri izardii denitrificans Brevibacterium ButtiauxellaBeutenbergia otitidis noackiae Beutenbergia Brevibacterium Buttiauxellacavernae oxydans warmboldiae Brevibacterium Butyrivibrio paucivoransButyrivibrio Brevibacterium fibrisolvens stationis Butyrivibrio hungateiButyrivibrio proteoclasticus Bacillus B. acidiceler B. aminovorans B.glucanolyticus B. taeanensis B. lautus B. acidicola B. amylolyticus B.gordonae B. tequilensis B. lehensis B. acidiproducens B. andreesenii B.gottheilii B. thermantarcticus B. lentimorbus B. acidocaldarius B.aneurinilyticus B. graminis B. thermoaerophilus B. lentus B.acidoterrestris B. anthracis B. halmapalus B. thermoamylovorans B.licheniformis B. aeolius B. aquimaris B. haloalkaliphilus B.thermocatenulatus B. ligniniphilus B. aerius B. arenosi B. halochares B.thermocloacae B. litoralis B. aerophilus B. arseniciselenatis B.halodenitrificans B. thermocopriae B. locisalis B. agaradhaerens B.arsenicus B. halodurans B. thermodenitrificans B. luciferensis B. agriB. aurantiacus B. halophilus B. thermoglucosidasius B. luteolus B.aidingensis B. arvi B. halosaccharovorans B. thermolactis B. luteus B.akibai B. aryabhattai B. hemicellulosilyticus B. thermoleovorans B.macauensis B. alcalophilus B. asahii B. hemicentroti B. thermophilus B.macerans B. algicola B. atrophaeus B. herbersteinensis B. thermoruber B.macquariensis B. alginolyticus B. axarquiensis B. horikoshii B.thermosphaericus B. macyae B. alkalidiazotrophicus B. azotofixans B.horneckiae B. thiaminolyticus B. malacitensis B. alkalinitrilicus B.azotoformans B. horti B. thioparans B. mannanilyticus B. alkalisediminisB. badius B. huizhouensis B. thuringiensis B. marisflavi B.alkalitelluris B. barbaricus B. humi B. tianshenii B. marismortui B.altitudinis B. bataviensis B. hwajinpoensis B. trypoxylicola B.marmarensis B. alveayuensis B. beijingensis B. idriensis B. tusciae B.massiliensis B. alvei B. benzoevorans B. indicus B. validus B.megaterium B. amyloliquefaciens B. beringensis B. infantis B.vallismortis B. mesonae B.a. subsp. amyloliquefaciens B. berkeleyi B.infernus B. vedderi B. methanolicus B.a. subsp. plantarum B. beveridgeiB. insolitus B. velezensis B. methylotrophicus B. dipsosauri B.bogoriensis B. invictae B. vietnamensis B. migulanus B. drentensis B.boroniphilus B. iranensis B. vireti B. mojavensis B. edaphicus B.borstelensis B. isabeliae B. vulcani B. mucilaginosus B. ehimensis B.brevis Migula B. isronensis B. wakoensis B. muralis B. eiseniae B.butanolivorans B. jeotgali B. weihenstephanensis B. murimartini B.enclensis B. canaveralius B. kaustophilus B. xiamenensis B. mycoides B.endophyticus B. carboniphilus B. kobensis B. xiaoxiensis B. naganoensisB. endoradicis B. cecembensis B. kochii B. zhanjiangensis B. nanhaiensisB. farraginis B. cellulosilyticus B. kokeshiiformis B. peoriae B.nanhaiisediminis B. fastidiosus B. centrosporus B. koreensis B.persepolensis B. nealsonii B. fengqiuensis B. cereus B. korlensis B.persicus B. neidei B. firmus B. chagannorensis B. kribbensis B. pervagusB. neizhouensis B. flexus B. chitinolyticus B. krulwichiae B.plakortidis B. niabensis B. foraminis B. chondroitinus B. laevolacticusB. pocheonensis B. niacini B. fordii B. choshinensis B. larvae B.polygoni B. novalis B. formosus B. chungangensis B. laterosporus B.polymyxa B. oceanisediminis B. fortis B. cibi B. salexigens B. popilliaeB. odysseyi B. fumarioli B. circulans B. saliphilus B. pseudalcalophilusB. okhensis B. funiculus B. clarkii B. schlegelii B. pseudofirmus B.okuhidensis B. fusiformis B. clausii B. sediminis B. pseudomycoides B.oleronius B. galactophilus B. coagulans B. selenatarsenatis B.psychrodurans B. oryzaecorticis B. galactosidilyticus B. coahuilensis B.selenitireducens B. psychrophilus B. oshimensis B. galliciensis B.cohnii B. seohaeanensis B. psychrosaccharolyticus B. pabuli B. gelatiniB. composti B. shacheensis B. psychrotolerans B. pakistanensis B.gibsonii B. curdlanolyticus B. shackletonii B. pulvifaciens B. pallidusB. ginsengi B. cycloheptanicus B. siamensis B. pumilus B. pallidus B.ginsengihumi B. cytotoxicus B. silvestris B. purgationiresistens B.panacisoli B. ginsengisoli B. daliensis B. simplex B. pycnus B.panaciterrae B. globisporus B. decisifrondis B. siralis B. qingdaonensisB. pantothenticus (eg, B.g. subsp. B. decolorationis B. smithii B.qingshengii B. parabrevis Globisporus; B. deserti B. soli B. reuszeri B.paraflexus or B.g. B. solimangrovi B. rhizosphaerae B. pasteurii subsp.Marinus) B. solisalsi B. rigui B. patagoniensis B. songklensis B. rurisB. sonorensis B. safensis B. sphaericus B. salarius B. sporothermoduransB. stearothermophilus B. stratosphericus B. subterraneus B. subtilis(eg, B.s. subsp. Inaquosorum; or B.s. subsp. Spizizeni; or B.s. subsp.Subtilis) Caenimonas Campylobacter Cardiobacterium CatenuloplanesCurtobacterium Caenimonas koreensis Campylobacter coli CardiobacteriumCatenuloplanes Curtobacterium Caldalkalibacillus Campylobacter hominisatrovinosus albidum Caldalkalibacillus concisus CarnimonasCatenuloplanes Curtobacterium uzonensis Campylobacter Carnimonascastaneus citreus Caldanaerobacter curvus nigrificans CatenuloplanesCaldanaerobacter Campylobacter fetus Carnobacterium crispus subterraneusCampylobacter Carnobacterium Catenuloplanes Caldanaerobius gracilisalterfunditum indicus Caldanaerobius Campylobacter CarnobacteriumCatenuloplanes fijiensis helveticus divergens japonicus CaldanaerobiusCampylobacter Carnobacterium Catenuloplanes polysaccharolyticus hominisfunditum nepalensis Caldanaerobius zeae Campylobacter CarnobacteriumCatenuloplanes Caldanaerovirga hyointestinalis gallinarum nigerCaldanaerovirga Campylobacter Carnobacterium Chryseobacteriumacetigignens jejuni maltaromaticum Chryseobacterium CaldicellulosiruptorCampylobacter lari Carnobacterium balustinum CaldicellulosiruptorCampylobacter mobile Citrobacter bescii mucosalis Carnobacterium C.amalonaticus Caldicellulosiruptor Campylobacter viridans C. braakiikristjanssonii rectus Caryophanon C. diversus CaldicellulosiruptorCampylobacter Caryophanon C. farmeri owensensis showae latum C. freundiiCampylobacter Caryophanon C. gillenii sputorum tenue C. koseriCampylobacter Catellatospora C. murliniae upsaliensis Catellatospora C.pasteurii ^([1]) Capnocytophaga citrea C. rodentium CapnocytophagaCatellatospora C. sedlakii canimorsus methionotrophica C. werkmaniiCapnocytophaga Catenococcus C. youngae cynodegmi CatenococcusClostridium Capnocytophaga thiocycli (see below) gingivalis CoccochlorisCapnocytophaga Coccochloris granulosa elabens CapnocytophagaCorynebacterium haemolytica Corynebacterium Capnocytophaga flavescensochracea Corynebacterium Capnocytophaga variabile sputigena ClostridiumClostridium absonum, Clostridium aceticum, Clostridium acetireducens,Clostridium acetobutylicum, Clostridium acidisoli, Clostridiumaciditolerans, Clostridium acidurici, Clostridium aerotolerans,Clostridium aestuarii, Clostridium akagii, Clostridium aldenense,Clostridium aldrichii, Clostridium algidicarni, Clostridiumalgidixylanolyticum, Clostridium algifaecis, Clostridium algoriphilum,Clostridium alkalicellulosi, Clostridium aminophilum, Clostridiumaminovalericum, Clostridium amygdalinum, Clostridium amylolyticum,Clostridium arbusti, Clostridium arcticum, Clostridium argentinense,Clostridium asparagiforme, Clostridium aurantibutyricum, Clostridiumautoethanogenum, Clostridium baratii, Clostridium barkeri, Clostridiumbartlettii, Clostridium beijerinckii, Clostridium bifermentans,Clostridium bolteae, Clostridium bornimense, Clostridium botulinum,Clostridium bowmanii, Clostridium bryantii, Clostridium butyricum,Clostridium cadaveris, Clostridium caenicola, Clostridiumcaminithermale, Clostridium carboxidivorans, Clostridium carnis,Clostridium cavendishii, Clostridium celatum, Clostridiumcelerecrescens, Clostridium cellobioparum, Clostridiumcellulofermentans, Clostridium cellulolyticum, Clostridium cellulosi,Clostridium cellulovorans, Clostridium chartatabidum, Clostridiumchauvoei, Clostridium chromiireducens, Clostridium citroniae,Clostridium clariflavum, Clostridium clostridioforme, Clostridiumcoccoides, Clostridium cochlearium, Clostridium colletant, Clostridiumcolicanis, Clostridium colinum, Clostridium collagenovorans, Clostridiumcylindrosporum, Clostridium difficile, Clostridium diolis, Clostridiumdisporicum, Clostridium drakei, Clostridium durum, Clostridiumestertheticum, Clostridium estertheticum estertheticum, Clostridiumestertheticum laramiense, Clostridium fallax, Clostridium felsineum,Clostridium fervidum, Clostridium fimetarium, Clostridiumformicaceticum, Clostridium frigidicarnis, Clostridium frigoris,Clostridium ganghwense, Clostridium gasigenes, Clostridium ghonii,Clostridium glycolicum, Clostridium glycyrrhizinilyticum, Clostridiumgrantii, Clostridium haemolyticum, Clostridium halophilum, Clostridiumhastiforme, Clostridium hathewayi, Clostridium herbivorans, Clostridiumhiranonis, Clostridium histolyticum, Clostridium homopropionicum,Clostridium huakuii, Clostridium hungatei, Clostridium hydrogeniformans,Clostridium hydroxybenzoicum, Clostridium hylemonae, Clostridiumjejuense, Clostridium indolis, Clostridium innocuum, Clostridiumintestinale, Clostridium irregulare, Clostridium isatidis, Clostridiumjosui, Clostridium kluyveri, Clostridium lactatifermentans, Clostridiumlacusfryxellense, Clostridium laramiense, Clostridium lavalense,Clostridium lentocellum, Clostridium lentoputrescens, Clostridiumleptum, Clostridium limosum, Clostridium litorale, Clostridiumlituseburense, Clostridium ljungdahlii, Clostridium lortetii,Clostridium lundense, Clostridium magnum, Clostridium malenominatum,Clostridium mangenotii, Clostridium mayombei, Clostridiummethoxybenzovorans, Clostridium methylpentosum, Clostridiumneopropionicum, Clostridium nexile, Clostridium nitrophenolicum,Clostridium novyi, Clostridium oceanicum, Clostridium orbiscindens,Clostridium oroticum, Clostridium oxalicum, Clostridium papyrosolvens,Clostridium paradoxum, Clostridium paraperfringens (Alias: C. welchii),Clostridium paraputrificum, Clostridium pascui, Clostridiumpasteurianum, Clostridium peptidivorans, Clostridium perenne,Clostridium perfringens, Clostridium pfennigii, Clostridiumphytofermentans, Clostridium piliforme, Clostridium polysaccharolyticum,Clostridium populeti, Clostridium propionicum, Clostridiumproteoclasticum, Clostridium proteolyticum, Clostridium psychrophilum,Clostridium puniceum, Clostridium purinilyticum, Clostridiumputrefaciens, Clostridium putrificum, Clostridium quercicolum,Clostridium quinii, Clostridium ramosum, Clostridium rectum, Clostridiumroseum, Clostridium saccharobutylicum, Clostridium saccharogumia,Clostridium saccharolyticum, Clostridium saccharoperbutylacetonicum,Clostridium sardiniense, Clostridium sartagoforme, Clostridiumscatologenes, Clostridium schirmacherense, Clostridium scindens,Clostridium septicum, Clostridium sordellii, Clostridium sphenoides,Clostridium spiroforme, Clostridium sporogenes, Clostridiumsporosphaeroides, Clostridium stercorarium, Clostridium stercorariumleptospartum, Clostridium stercorarium stercorarium, Clostridiumstercorarium thermolacticum, Clostridium sticklandii, Clostridiumstraminisolvens, Clostridium subterminale, Clostridium sufflavum,Clostridium sulfidigenes, Clostridium symbiosum, Clostridium tagluense,Clostridium tepidiprofundi, Clostridium termitidis, Clostridium tertium,Clostridium tetani, Clostridium tetanomorphum, Clostridiumthermaceticum, Clostridium thermautotrophicum, Clostridiumthermoalcaliphilum, Clostridium thermobutyricum, Clostridiumthermocellum, Clostridium thermocopriae, Clostridiumthermohydrosulfuricum, Clostridium thermolacticum, Clostridiumthermopalmarium, Clostridium thermopapyrolyticum, Clostridiumthermosaccharolyticum, Clostridium thermosuccinogenes, Clostridiumthermosulfurigenes, Clostridium thiosulfatireducens, Clostridiumtyrobutyricum, Clostridium uliginosum, Clostridium ultunense,Clostridium villosum, Clostridium vincentii, Clostridium viride,Clostridium xylanolyticum, Clostridium xylanovorans DactylosporangiumDeinococcus Delftia Echinicola Dactylosporangium Deinococcus DelftiaEchinicola aurantiacum aerius acidovorans pacifica DactylosporangiumDeinococcus Desulfovibrio Echinicola fulvum apachensis Desulfovibriovietnamensis Dactylosporangium Deinococcus desulfuricans matsuzakienseaquaticus Diplococcus Dactylosporangium Deinococcus Diplococcus roseumaquatilis pneumoniae Dactylosporangium Deinococcus caeni thailandenseDeinococcus Dactylosporangium radiodurans vinaceum Deinococcusradiophilus Enterobacter Enterobacter kobei FaecalibacteriumFlavobacterium E. aerogenes E. ludwigii Faecalibacterium FlavobacteriumE. amnigenus E. mori prausnitzii antarcticum E. agglomerans E.nimipressuralis Fangia Flavobacterium E. arachidis E. oryzae Fangiaaquatile E. asburiae E. pulveris hongkongensis Flavobacterium E.cancerogenous E. pyrinus Fastidiosipila aquidurense E. cloacae E.radicincitans Fastidiosipila Flavobacterium E. cowanii E. tayloraesanguinis balustinum E. dissolvens E. turicensis FusobacteriumFlavobacterium E. gergoviae E. sakazakii Fusobacterium croceum E.helveticus Enterobacter soli nucleatum Flavobacterium E. hormaecheiEnterococcus cucumis E. intermedius Enterococcus Flavobacterium duransdaejeonense Enterococcus Flavobacterium faecalis defluvii EnterococcusFlavobacterium faecium degerlachei Erwinia Flavobacterium Erwiniahapontici denitrificans Escherichia Flavobacterium Escherichia colifilum Flavobacterium flevense Flavobacterium frigidarium Flavobacteriummizutaii Flavobacterium okeanokoites Gaetbulibacter HaemophilusIdeonella Janibacter Gaetbulibacter Haemophilus Ideonella Janibactersaemankumensis aegyptius azotifigens anophelis GallibacteriumHaemophilus Idiomarina Janibacter Gallibacterium anatis aphrophilusIdiomarina corallicola Gallicola Haemophilus felis abyssalis JanibacterGallicola barnesae Haemophilus Idiomarina limosus Garciella gallinarumbaltica Janibacter Garciella Haemophilus Idiomarina melonisnitratireducens haemolyticus fontislapidosi Janibacter GeobacillusHaemophilus Idiomarina terrae Geobacillus influenzae loihiensisJannaschia thermoglucosidasius Haemophilus Idiomarina JannaschiaGeobacillus paracuniculus ramblicola cystaugens stearothermophilusHaemophilus Idiomarina Jannaschia Geobacter parahaemolyticus seosinensishelgolandensis Geobacter Haemophilus Idiomarina Jannaschia bemidjiensisparainfluenzae zobellii pohangensis Geobacter bremensis HaemophilusIgnatzschineria Jannaschia Geobacter chapellei paraphrohaemolyticusIgnatzschineria rubra Geobacter grbiciae Haemophilus larvaeJanthinobacterium Geobacter parasuis Ignavigranum Janthinobacteriumhydrogenophilus Haemophilus Ignavigranum agaricidamnosum Geobacterlovleyi pittmaniae ruoffiae Janthinobacterium Geobacter HafniaIlumatobacter lividum metallireducens Hafnia alvei Ilumatobacter JejuiaGeobacter pelophilus Hahella fluminis Jejuia Geobacter pickeringiiHahella Ilyobacter pallidilutea Geobacter ganghwensis IlyobacterJeotgalibacillus sulfurreducens Halalkalibacillus delafieldiiJeotgalibacillus Geodermatophilus Halalkalibacillus Ilyobacteralimentarius Geodermatophilus halophilus insuetus Jeotgalicoccusobscurus Helicobacter Ilyobacter Jeotgalicoccus GluconacetobacterHelicobacter polytropus halotolerans Gluconacetobacter pylori Ilyobacterxylinus tartaricus Gordonia Gordonia rubripertincta Kaistia LabedellaListeria ivanovii Micrococcus Nesterenkonia Kaistia adipata Labedella L.marthii Micrococcus Nesterenkonia Kaistia soli gwakjiensis L.monocytogenes luteus holobia Kangiella Labrenzia L. newyorkensisMicrococcus Nocardia Kangiella Labrenzia L. riparia lylae Nocardiaaquimarina aggregata L. rocourtiae Moraxella argentinensis Kangiellakoreensis Labrenzia alba L. seeligeri Moraxella bovis NocardiaKerstersia Labrenzia L. weihenstephanensis Moraxella corallinaKerstersia gyiorum alexandrii L. welshimeri nonliquefaciens NocardiaKiloniella Labrenzia marina Listonella Moraxella otitidiscaviarumKiloniella laminariae Labrys Listonella osloensis Klebsiella Labrysanguillarum Nakamurella K. granulomatis methylaminiphilus MacrococcusNakamurella K. oxytoca Labrys Macrococcus multipartita K. pneumoniaemiyagiensis bovicus Nannocystis K. terrigena Labrys monachusMarinobacter Nannocystis K. variicola Labrys Marinobacter pusillaKluyvera okinawensis algicola Natranaerobius Kluyvera ascorbata LabrysMarinobacter Natranaerobius Kocuria portucalensis bryozoorumthermophilus Kocuria roasea Lactobacillus Marinobacter NatranaerobiusKocuria varians [see below] flavimaris trueperi Kurthia LaceyellaMeiothermus Naxibacter Kurthia zopfii Laceyella putida MeiothermusNaxibacter Lechevalieria ruber alkalitolerans LechevalieriaMethylophilus Neisseria aerocolonigenes Methylophilus Neisseria cinereaLegionella methylotrophus Neisseria [see below] Microbacteriumdenitrificans Listeria Microbacterium Neisseria L. aquaticaammoniaphilum gonorrhoeae L. booriae Microbacterium Neisseria L.cornellensis arborescens lactamica L. fleischmannii MicrobacteriumNeisseria mucosa L. floridensis liquefaciens Neisseria sicca L.grandensis Microbacterium Neisseria subflava L. grayi oxydansNeptunomonas L. innocua Neptunomonas japonica Lactobacillus L.acetotolerans L. catenaformis L. mali L. parakefiri L. sakei L.acidifarinae L. ceti L. manihotivorans L. paralimentarius L. salivariusL. acidipiscis L. coleohominis L. mindensis L. paraplantarum L.sanfranciscensis L. acidophilus L. collinoides L. mucosae L. pentosus L.satsumensis Lactobacillus agilis L. composti L. murinus L. perolens L.secaliphilus L. algidus L. concavus L. nagelii L. plantarum L. sharpeaeL. alimentarius L. coryniformis L. namurensis L. pontis L. siliginis L.amylolyticus L. crispatus L. nantensis L. protectus L. spicheri L.amylophilus L. crustorum L. oligofermentans L. psittaci L. suebicus L.amylotrophicus L. curvatus L. oris L. rennini L. thailandensis L.amylovorus L. delbrueckii L. panis L. reuteri L. ultunensis L. animalissubsp. bulgaricus L. pantheris L. rhamnosus L. vaccinostercus L. antriL. delbrueckii L. parabrevis L. rimae L. vaginalis L. apodemi subsp.delbrueckii L. parabuchneri L. rogosae L. versmoldensis L. aviarius L.delbrueckii L. paracasei L. rossiae L. vini L. bifermentans subsp.lactis L. paracollinoides L. ruminis L. vitulinus L. brevis L.dextrinicus L. parafarraginis L. saerimneri L. zeae L. buchneri L.diolivorans L. homohiochii L. jensenii L. zymae L. camelliae L. equi L.iners L. johnsonii L. gastricus L. casei L. equigenerosi L. ingluviei L.kalixensis L. ghanensis L. kitasatonis L. farraginis L. intestinalis L.kefiranofaciens L. graminis L. kunkeei L. farciminis L. fuchuensis L.kefiri L. hammesii L. leichmannii L. fermentum L. gallinarum L. kimchiiL. hamsteri L. lindneri L. fornicalis L. gasseri L. helveticus L.harbinensis L. malefermentans L. fructivorans L. hilgardii L.hayakitensis L. frumenti Legionella Legionella Legionella CandidatusLegionella Legionella adelaidensis drancourtii jeonii quinlivaniiLegionella anisa Legionella Legionella Legionella Legionelladresdenensis jordanis rowbothamii beliardensis Legionella LegionellaLegionella Legionella drozanskii lansingensis rubrilucensbirminghamensis Legionella Legionella Legionella Legionella dumoffiilondiniensis sainthelensi bozemanae Legionella erythra LegionellaLegionella Legionella brunensis Legionella longbeachae santicrucisLegionella fairfieldensis Legionella lytica Legionella busanensisLegionella fallonii Legionella shakespearei Legionella cardiacaLegionella feeleii maceachernii Legionella Legionella cherrii LegionellaLegionella spiritensis Legionella geestiana massiliensis Legionellacincinnatiensis Legionella Legionella steelei Legionella genomospeciesmicdadei Legionella clemsonensis Legionella Legionella steigerwaltiiLegionella gormanii monrovica Legionella donaldsonii LegionellaLegionella taurinensis gratiana moravica Legionella LegionellaLegionella tucsonensis gresilensis nagasakiensis Legionella LegionellaLegionella tunisiensis hackeliae nautarum Legionella LegionellaLegionella wadsworthii impletisoli norrlandica Legionella LegionellaLegionella waltersii israelensis oakridgensis Legionella LegionellaLegionella worsleiensis jamestowniensis parisiensis LegionellaLegionella yabuuchiae pittsburghensis Legionella pneumophila Legionellaquateirensis Oceanibulbus Paenibacillus Prevotella QuadrisphaeraOceanibulbus Paenibacillus Prevotella Quadrisphaera indolifexthiaminolyticus albensis granulorum Oceanicaulis Pantoea Prevotellaamnii Quatrionicoccus Oceanicaulis Pantoea Prevotella Quatrionicoccusalexandrii agglomerans bergensis australiensis Oceanicola ParacoccusPrevotella bivia Quinella Oceanicola batsensis Paracoccus Prevotellabrevis Quinella ovalis Oceanicola alcaliphilus Prevotella Ralstoniagranulosus Paucimonas bryantii Ralstonia Oceanicola PaucimonasPrevotella buccae eutropha nanhaiensis lemoignei Prevotella RalstoniaOceanimonas Pectobacterium buccalis insidiosa Oceanimonas PectobacteriumPrevotella copri Ralstonia baumannii aroidearum Prevotellamannitolilytica Oceaniserpentilla Pectobacterium dentalis RalstoniaOceaniserpentilla atrosepticum Prevotella pickettii haliotisPectobacterium denticola Ralstonia Oceanisphaera betavasculorumPrevotella disiens pseudosolanacearum Oceanisphaera PectobacteriumPrevotella Ralstonia syzygii donghaensis cacticida histicola RalstoniaOceanisphaera Pectobacterium Prevotella solanacearum litoraliscarnegieana intermedia Ramlibacter Oceanithermus PectobacteriumPrevotella Ramlibacter Oceanithermus carotovorum maculosa henchirensisdesulfurans Pectobacterium Prevotella Ramlibacter Oceanithermuschrysanthemi marshii tataouinensis profundus Pectobacterium PrevotellaRaoultella Oceanobacillus cypripedii melaninogenica RaoultellaOceanobacillus caeni Pectobacterium Prevotella micans ornithinolyticaOceanospirillum rhapontici Prevotella Raoultella OceanospirillumPectobacterium multiformis planticola linum wasabiae PrevotellaRaoultella Planococcus nigrescens terrigena Planococcus Prevotellaoralis Rathayibacter citreus Prevotella oris RathayibacterPlanomicrobium Prevotella caricis Planomicrobium oulorum Rathayibacterokeanokoites Prevotella pallens festucae Plesiomonas Prevotella salivaeRathayibacter Plesiomonas Prevotella iranicus shigelloides stercoreaRathayibacter Proteus Prevotella rathayi Proteus vulgaris tanneraeRathayibacter Prevotella toxicus timonensis Rathayibacter Prevotellatritici veroralis Rhodobacter Providencia Rhodobacter Providenciasphaeroides stuartii Ruegeria Pseudomonas Ruegeria Pseudomonasgelatinovorans aeruginosa Pseudomonas alcaligenes Pseudomonasanguillispetica Pseudomonas fluorescens Pseudoalteromonas haloplanktisPseudomonas mendocina Pseudomonas pseudoalcaligenes Pseudomonas putidaPseudomonas tutzeri Pseudomonas syringae Psychrobacter Psychrobacterfaecalis Psychrobacter phenylpyruvicus Saccharococcus SagittulaSanguibacter Stenotrophomonas Tatlockia Saccharococcus Sagittulastellata Sanguibacter Stenotrophomonas Tatlockia thermophilusSalegentibacter keddieii maltophilia maceachernii SaccharomonosporaSalegentibacter Sanguibacter Streptococcus Tatlockia Saccharomonosporasalegens suarezii [also see below] micdadei azurea SalimicrobiumSaprospira Streptomyces Tenacibaculum Saccharomonospora SalimicrobiumSaprospira Streptomyces Tenacibaculum cyanea album grandis achromogenesamylolyticum Saccharomonospora Salinibacter Sarcina StreptomycesTenacibaculum viridis Salinibacter ruber Sarcina maxima cesalbusdiscolor Saccharophagus Salinicoccus Sarcina ventriculi StreptomycesTenacibaculum Saccharophagus Salinicoccus Sebaldella cescaepitosusgallaicum degradans alkaliphilus Sebaldella Streptomyces TenacibaculumSaccharopolyspora Salinicoccus termitidis cesdiastaticus lutimarisSaccharopolyspora hispanicus Serratia Streptomyces Tenacibaculumerythraea Salinicoccus roseus Serratia fonticola cesexfoliatusmesophilum Saccharopolyspora Salinispora Serratia StreptomycesTenacibaculum gregorii Salinispora marcescens fimbriatus skagerrakenseSaccharopolyspora arenicola Sphaerotilus Streptomyces Tepidanaerobacterhirsuta Salinispora tropica Sphaerotilus fradiae TepidanaerobacterSaccharopolyspora Salinivibrio natans Streptomyces syntrophicus hordeiSalinivibrio Sphingobacterium fulvissimus Tepidibacter Saccharopolysporacosticola Sphingobacterium Streptomyces Tepidibacter rectivirgulaSalmonella multivorum griseoruber formicigenes SaccharopolysporaSalmonella bongori Staphylococcus Streptomyces Tepidibacter spinosaSalmonella enterica [see below] griseus thalassicus SaccharopolysporaSalmonella Streptomyces Thermus taberi subterranea lavendulae ThermusSaccharothrix Salmonella typhi Streptomyces aquaticus Saccharothrixphaeochromogenes Thermus australiensis Streptomyces filiformisSaccharothrix thermodiastaticus Thermus coeruleofusca Streptomycesthermophilus Saccharothrix tubercidicus espanaensis Saccharothrixlongispora Saccharothrix mutabilis Saccharothrix syringae Saccharothrixtangerinus Saccharothrix texasensis Staphylococcus S. arlettae S.equorum S. microti S. schleiferi S. agnetis S. felis S. muscae S. sciuriS. aureus S. fleurettii S. nepalensis S. simiae S. auricularis S.gallinarum S. pasteuri S. simulans S. capitis S. haemolyticus S.petrasii S. stepanovicii S. caprae S. hominis S. pettenkoferi S.succinus S. carnosus S. hyicus S. piscifermentans S. vitulinus S.caseolyticus S. intermedius S. pseudintermedius S. warneri S.chromogenes S. kloosii S. pseudolugdunensis S. xylosus S. cohnii S. leeiS. pulvereri S. condimenti S. lentus S. rostri S. delphini S.lugdunensis S. saccharolyticus S. devriesei S. lutrae S. saprophyticusS. epidermidis S. lyticans S. massiliensis Streptococcus StreptococcusStreptococcus Streptococcus Streptococcus agalactiae infantariusorisratti thermophilus Streptococcus anginosus Streptococcus iniaeStreptococcus Streptococcus Streptococcus bovis Streptococcusparasanguinis sanguinis Streptococcus canis intermedius StreptococcusStreptococcus Streptococcus Streptococcus peroris sobrinus constellatuslactarius Streptococcus Streptococcus Streptococcus downei Streptococcuspneumoniae suis Streptococcus milleri Streptococcus Streptococcusdysgalactiae Streptococcus mitis pseudopneumoniae uberis Streptococcusequines Streptococcus Streptococcus Streptococcus Streptococcus faecalismutans pyogenes vestibularis Streptococcus ferus Streptococcus oralisStreptococcus Streptococcus Streptococcus ratti viridans tigurinusStreptococcus Streptococcus salivariu zooepidemicus UliginosibacteriumVagococcus Vibrio Virgibacillus Xanthobacter UliginosibacteriumVagococcus Vibrio aerogenes Virgibacillus Xanthobacter gangwonensecarniphilus Vibrio halodenitrificans agilis Ulvibacter Vagococcusaestuarianus Virgibacillus Xanthobacter Ulvibacter litoralis elongatusVibrio albensis pantothenticus aminoxidans Umezawaea Vagococcus fessusVibrio Weissella Xanthobacter Umezawaea tangerina Vagococcus fluvialisalginolyticus Weissella cibaria autotrophicus Undibacterium Vagococcuslutrae Vibrio campbellii Weissella confusa Xanthobacter Undibacteriumpigrum Vagococcus Vibrio cholerae Weissella flavus Ureaplasmasalmoninarum Vibrio halotolerans Xanthobacter Ureaplasma Variovoraxcincinnatiensis Weissella tagetidis urealyticum Variovorax Vibriohellenica Xanthobacter Ureibacillus boronicumulans coralliilyticusWeissella viscosus Ureibacillus composti Variovorax Vibrio kandleriXanthomonas Ureibacillus dokdonensis cyclitrophicus WeissellaXanthomonas suwonensis Variovorax Vibrio koreensis albilineansUreibacillus terrenus paradoxus diazotrophicus Weissella minorXanthomonas Ureibacillus Variovorax soli Vibrio fluvialis Weissellaalfalfae thermophilus Veillonella Vibrio furnissii paramesenteroidesXanthomonas Ureibacillus Veillonella atypica Vibrio gazogenes Weissellasoli arboricola thermosphaericus Veillonella caviae Vibrio halioticoliWeissella Xanthomonas Veillonella criceti Vibrio harveyi thailandensisaxonopodis Veillonella dispar Vibrio Weissella Xanthomonas Veillonellaichthyoenteri viridescens campestris montpellierensis Vibrio WilliamsiaXanthomonas Veillonella parvula mediterranei Williamsia citriVeillonella ratti Vibrio marianensis Xanthomonas Veillonellametschnikovii Williamsia maris codiaei rodentium Vibrio mytiliWilliamsia Xanthomonas Venenivibrio Vibrio natriegens serinedenscucurbitae Venenivibrio Vibrio Winogradskyella Xanthomonasstagnispumantis navarrensis Winogradskyella euvesicatoriaVerminephrobacter Vibrio nereis thalassocola XanthomonasVerminephrobacter Vibrio Wolbachia fragariae eiseniae nigripulchritudoWolbachia Xanthomonas Verrucomicrobium Vibrio ordalii persica fuscansVerrucomicrobium Vibrio orientalis Wolinella Xanthomonas spinosum VibrioWolinella gardneri parahaemolyticus succinogenes Xanthomonas Vibriopectenicida Zobellia hortorum Vibrio penaeicida Zobellia XanthomonasVibrio galactanivorans hyacinthi proteolyticus Zobellia uliginosaXanthomonas Vibrio shilonii Zoogloea perforans Vibrio splendidusZoogloea Xanthomonas Vibrio tubiashii ramigera phaseoli Vibriovulnificus Zoogloea Xanthomonas resiniphila pisi Xanthomonas populiXanthomonas theicola Xanthomonas translucens Xanthomonas vesicatoriaXylella Xylella fastidiosa Xylophilus Xylophilus ampelinus XenophilusYangia Yersinia Zooshikella Zobellella Xenophilus azovorans Yangiapacifica mollaretii Zooshikella Zobellella Xenorhabdus Yaniella Yersiniaganghwensis denitrificans Xenorhabdus beddingii Yaniella flavaphilomiragia Zunongwangia Zobellella Xenorhabdus bovienii YaniellaYersinia pestis Zunongwangia taiwanensis Xenorhabdus halotoleransYersinia profunda Zeaxanthinibacter cabanillasii Yeosuanapseudotuberculosis Zymobacter Zeaxanthinibacter Xenorhabdus doucetiaeYeosuana Yersinia rohdei Zymobacter enoshimensis Xenorhabdus griffiniaearomativorans Yersinia ruckeri palmae Zhihengliuella Xenorhabdushominickii Yersinia Yokenella Zymomonas Zhihengliuella XenorhabdusYersinia aldovae Yokenella Zymomonas halotolerans koppenhoeferi Yersiniabercovieri regensburgei mobilis Xylanibacterium Xenorhabdus YersiniaYonghaparkia Zymophilus Xylanibacterium nematophila enterocoliticaYonghaparkia Zymophilus ulmi Xenorhabdus poinarii Yersinia alkaliphilapaucivorans Xylanibacter entomophaga Zavarzinia Zymophilus Xylanibacteroryzae Yersinia Zavarzinia raffinosivorans frederiksenii compransorisYersinia intermedia Yersinia kristensenii

TABLE 2 MEDICAMENTS ACE inhibitors with calcium decongestantsrespiratory agents otic agents channel blocking agents dermatologicalagents sex hormones renin inhibitors ACE inhibitors with thiazidesdiagnostic topical agents respiratory agents adamantane antiviralsradiopharmaceuticals uncategorized respiratory inhalant adrenal corticalsteroids diarylquinolines agents products adrenal corticosteroiddibenzazepine vaginal agents rifamycin derivatives inhibitorsanticonvulsants mitotic inhibitors salicylates adrenergicbronchodilators digestive enzymes monoamine oxidase sclerosing agentsagents for hypertensive dipeptidyl peptidase 4 inhibitors secondgeneration emergencies inhibitors mouth and throat cephalosporins agentsfor pulmonary diuretics products selective estrogen hypertensiondopaminergic mTOR inhibitors receptor modulators aldosterone receptorantiparkinsonism agents mucolytics selective antagonists drugs used inalcohol multikinase immunosuppressants alkylating agents dependenceinhibitors selective allergenics echinocandins muscle relaxantsphosphodiesterase-4 alpha-glucosidase inhibitors EGFR inhibitorsmydriatics inhibitors alternative medicines estrogen receptor narcoticanalgesic selective serotonin amebicides antagonists combinationsreuptake inhibitors aminoglycosides estrogens narcotic analgesicsserotonin- aminopenicillins expectorants nasal anti-infectivesnorepinephrine aminosalicylates factor Xa inhibitors nasalantihistamines reuptake inhibitors AMPA receptor antagonists fatty acidderivative and decongestants serotoninergic amylin analogsanticonvulsants nasal lubricants and neuroenteric analgesic combinationsfibric acid derivatives irrigations modulators analgesics firstgeneration nasal preparations sex hormone androgens and anaboliccephalosporins nasal steroids combinations steroids fourth generationnatural penicillins sex hormones angiotensin converting cephalosporinsneprilysin inhibitors SGLT-2 inhibitors enzyme inhibitors functionalbowel neuraminidase skeletal muscle angiotensin II inhibitors withdisorder agents inhibitors relaxant combinations calcium channelblockers gallstone solubilizing neuromuscular skeletal muscleangiotensin II inhibitors with agents blocking agents relaxantsthiazides gamma-aminobutyric neuronal potassium smoking cessationangiotensin receptor blockers acid analogs channel openers agentsangiotensin receptor blockers gamma-aminobutyric next generationsomatostatin and and neprilysin inhibitors acid reuptake inhibitorscephalosporins somatostatin analogs anorectal preparationsgastrointestinal agents nicotinic acid spermicides anorexiants generalanesthetics derivatives statins antacids genitourinary tract NK1receptor sterile irrigating anthelmintics agents antagonists solutionsanti-angiogenic ophthalmic GI stimulants NNRTIs streptomyces agentsglucocorticoids non-cardioselective derivatives anti-CTLA-4 monoclonalglucose elevating beta blockers succinimide antibodies agentsnon-iodinated anticonvulsants anti-infectives glycopeptide antibioticscontrast media sulfonamides Anti-PD-1 monoclonal glycoprotein plateletnon-ionic iodinated sulfonylureas antibodies inhibitors contrast mediasynthetic ovulation antiadrenergic agents (central) glycylcyclinesnon-sulfonylureas stimulants with thiazides gonadotropin releasingnonsteroidal anti- tetracyclic antiadrenergic agents hormonesinflammatory antidepressants (peripheral) with thiazidesgonadotropin-releasing agents tetracyclines antiadrenergic agents,hormone antagonists NS5A inhibitors therapeutic centrally actinggonadotropins nucleoside reverse radiopharmaceuticals antiadrenergicagents, group I antiarrhythmics transcriptase therapeutic vaccinesperipherally acting group II inhibitors (NRTIs) thiazide diureticsantiandrogens antiarrhythmics nutraceutical thiazolidinedionesantianginal agents group III products thioxanthenes antiarrhythmicagents antiarrhythmics nutritional products third generationantiasthmatic combinations group IV ophthalmic cephalosporinsantibiotics/antineoplastics antiarrhythmics anesthetics thrombininhibitors anticholinergic antiemetics group V ophthalmic anti-thrombolytics anticholinergic antiparkinson antiarrhythmics infectivesthyroid drugs agents growth hormone ophthalmic anti- TNF alfa inhibitorsanticholinergic receptor blockers inflammatory tocolytic agentsbronchodilators growth hormones agents topical acne agentsanticholinergic chronotropic guanylate cyclase-C ophthalmic topicalagents agents agonists antihistamines and topical anestheticsanticholinergics/ H. pylori eradication decongestants topicalanti-infectives antispasmodics agents ophthalmic topical anti-rosaceaanticoagulant reversal agents H2 antagonists diagnostic agents agentsanticoagulants hedgehog pathway ophthalmic topical antibioticsanticonvulsants inhibitors glaucoma agents topical antifungalsantidepressants hematopoietic stem cell ophthalmic topicalantihistamines antidiabetic agents mobilizer lubricants and topicalantineoplastics antidiabetic combinations heparin antagonistsirrigations topical antipsoriatics antidiarrheals heparins ophthalmictopical antivirals antidiuretic hormones HER2 inhibitors preparationstopical astringents antidotes herbal products ophthalmic steroidstopical debriding antiemetic/antivertigo agents histone deacetylaseophthalmic steroids agents antifungals inhibitors with anti-infectivestopical depigmenting antigonadotropic agents hormones ophthalmicsurgical agents antigout agents hormones/ agents topical emollientsantihistamines antineoplastics oral nutritional topical keratolyticsantihyperlipidemic agents hydantoin supplements topical non-steroidalantihyperlipidemic anticonvulsants immunostimulants anti-inflammatoriescombinations hydrazide derivatives immunosuppressants topicalantihypertensive combinations immune globulins photochemotherapeuticsantihyperuricemic agents immunologic agents otic anestheticsantimalarial agents immunostimulants otic anti-infectives topicalrubefacient antimalarial combinations immunosuppressive oticpreparations topical steroids antimalarial quinolines agents oticsteroids topical steroids with antimetabolites impotence agents oticsteroids with anti-infectives antimigraine agents in vivo diagnosticanti-infectives triazine antineoplastic detoxifying biologicalsoxazolidinedione anticonvulsants agents incretin mimeticsanticonvulsants tricyclic antineoplastic interferons inhaledanti-infectives oxazolidinone antidepressants antineoplastics inhaledcorticosteroids antibiotics trifunctional antiparkinson agents inotropicagents parathyroid monoclonal antibodies antiplatelet agents insulinhormone and ultrasound contrast antipseudomonal penicillins insulin-likegrowth analogs media antipsoriatics factor PARP inhibitors upperrespiratory antipsychotics integrase strand transfer PCSK9 inhibitorscombinations antirheumatics inhibitor penicillinase urea anticonvulsantsantiseptic and germicides interferons resistant penicillins urea cycledisorder antithyroid agents interleukin inhibitors penicillins agentsantitoxins and antivenins interleukins peripheral opioid urinaryanti-infectives antituberculosis agents intravenous nutritional receptorantagonists urinary antispasmodics antituberculosis combinationsproducts peripheral opioid urinary pH modifiers antitussives iodinatedcontrast receptor mixed uterotonic agents antiviral agents mediaagonists/antagonists vaccine combinations antiviral boosters ioniciodinated contrast peripheral vaginal anti-infectives antiviralcombinations media vasodilators vaginal preparations antiviralinterferons iron products peripherally acting vasodilators anxiolytics,sedatives, and ketolides antiobesity agents vasopressin hypnoticslaxatives phenothiazine antagonists aromatase inhibitors leprostaticsantiemetics vasopressors atypical antipsychotics leukotriene modifiersphenothiazine VEGF/VEGFR azole antifungals lincomycin derivativesantipsychotics inhibitors bacterial vaccines local injectablephenylpiperazine viral vaccines barbiturate anticonvulsants anestheticsantidepressants viscosupplementation barbiturates local injectablephosphate binders agents BCR-ABL tyrosine kinase anesthetics with plasmaexpanders vitamin and mineral inhibitors corticosteroids plateletaggregation combinations benzodiazepine loop diuretics inhibitorsvitamins anticonvulsants lung surfactants platelet-stimulating5-alpha-reductase benzodiazepines lymphatic staining agents inhibitorsbeta blockers with calcium agents polyenes 5-aminosalicylates channelblockers lysosomal enzymes potassium sparing 5HT3 receptor beta blockerswith thiazides macrolide derivatives diuretics with antagonistsbeta-adrenergic blocking macrolides thiazides chloride channel agentsmagnetic resonance potassium-sparing activators beta-lactamaseinhibitors imaging contrast media diuretics cholesterol absorption bileacid sequestrants mast cell stabilizers probiotics inhibitorsbiologicals medical gas progesterone cholinergic agonistsbisphosphonates meglitinides receptor modulators cholinergic muscle bonemorphogenetic proteins metabolic agents progestins stimulants boneresorption inhibitors methylxanthines prolactin inhibitorscholinesterase bronchodilator combinations mineralocorticoidsprostaglandin D2 inhibitors bronchodilators minerals and antagonists CNSstimulants calcimimetics electrolytes protease inhibitors coagulationmodifiers calcineurin inhibitors agents protease-activated colonystimulating calcitonin analgesics receptor-1 factors calcium channelblocking antibiotics antagonists contraceptives agents anticonvulsantsproteasome corticotropin carbamate anticonvulsants antidepressantsinhibitors coumarins and carbapenems antidiabetic agents proton pumpindandiones carbonic anhydrase inhibitors antiemetics inhibitors cox-2inhibitors antifungals psoralens antihyperlipidemic psychotherapeuticagents agents antihypertensive psychotherapeutic combinationscombinations antimalarials purine nucleosides antineoplasticspyrrolidine antiparkinson agents anticonvulsants antipsychotic agentsquinolones antituberculosis agents radiocontrast agents antiviralsradiologic adjuncts anxiolytics, sedatives radiologic agents andhypnotics radiologic bone resorption conjugating agents inhibitorsradiopharmaceuticals cardiovascular agents central nervous systemrecombinant human agents erythropoietins coagulation modifiersanticonvulsants diagnostic dyes carbonic anhydrase diuretics inhibitorsgenitourinary tract cardiac stressing agents agents GI agentscardioselective beta hormones blockers metabolic agents cardiovascularophthalmic agents agents catecholamines CD20 monoclonal antibodies CD30monoclonal antibodies CD33 monoclonal antibodies CD38 monoclonalantibodies CD52 monoclonal antibodies central nervous system agentscephalosporins cephalosporins/beta- lactamase inhibitors cerumenolyticsCFTR combinations CFTR potentiators chelating agents chemokine receptorantagonist

The invention claimed is:
 1. A method of treating or reducing theprogression of an infection that is associated with sepsis or septicshock and is caused by pathogenic host cells of Escherichia coli,Staphylococcus, Streptococcus, Mycobacterium, Haemophilus or Pseudomonasin a human or animal subject, the method comprising selective targetingof the pathogenic host cells, wherein the host cells are comprised by amicrobiota in the subject, wherein the method comprising: a. contactingthe microbiota with an engineered nucleic acid sequence for producing ahost modifying (HM) crRNA, and b. producing the HM-crRNA in a host cell,wherein the HM-crRNA is operable with a Cas nuclease in the host cell toform a HM-CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas system, and wherein the HM-crRNA comprises a sequence thatis capable of hybridizing to a target sequence of the host cell to guidethe Cas nuclease to the target sequence in the host cell, whereby thetarget sequence is modified by the HM-CRISPR/Cas system and the hostcell is killed or growth of the host cell is reduced, wherein themicrobiota comprises a second species of bacteria that is different fromthe species of the pathogenic host cells, and wherein growth of thesecond species of bacteria is not substantially reduced, therebyreducing the proportion of host cells in the microbiota.
 2. The methodof claim 1, wherein the microbiota is selected from the group consistingof a gut microbiota, skin microbiota, oral cavity microbiota, throatmicrobiota, hair microbiota, armpit microbiota, rectal microbiota, analmicrobiota, ocular microbiota, nasal microbiota, tongue microbiota,liver microbiota, scrotal microbiota, mammary gland microbiota, earmicrobiota, organ microbiota and dental microbiota.
 3. The method ofclaim 1, wherein the microbiota is a kidney microbiota, urethramicrobiota, vaginal microbiota or penile microbiota.
 4. The method ofclaim 1, wherein the microbiota is a kidney microbiota.
 5. The method ofclaim 1, wherein the microbiota is a urethra microbiota.
 6. The methodof claim 1, wherein the microbiota is a lung microbiota.
 7. The methodof claim 1, wherein the Cas is a Cas3, Cas9 or Cpf1.
 8. The method ofclaim 1, wherein the engineered nucleic acid sequence is administeredintravenously to the subject.
 9. The method of claim 2, wherein theengineered nucleic acid sequence is administered intravenously to thesubject.
 10. The method of claim 6, wherein the engineered nucleic acidsequence is administered by inhalation to the subject.
 11. The method ofclaim 1, wherein each HM-crRNA is comprised by a single guide RNA(sgRNA).
 12. The method of claim 1, wherein the nucleic acid sequenceencoding the HM-crRNA is under a constitutive promoter.
 13. The methodof claim 1, wherein the Cas is a wild-type host cell endogenous Cas3and/or CASCADE Cas.
 14. The method of claim 1, wherein the nucleic acidsequence is comprised by a vector, wherein the vector is a virus, phage,prophage or conjugative plasmid.
 15. A method of treating or reducingthe progression of an infection that is associated with sepsis or septicshock and is caused by pathogenic host cells of Escherichia coli in ahuman or animal subject, the method comprising selective targeting ofthe pathogenic host cells, wherein the host cells are comprised by amicrobiota in the subject, wherein the method comprising: a. contactingthe microbiota with an engineered nucleic acid sequence for producing ahost modifying (HM) crRNA, and b. producing the HM-crRNA in a host cell,wherein the HM-crRNA is operable with a Cas nuclease in the host cell toform a HM-CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas system, and wherein the HM-crRNA comprises a sequence thatis capable of hybridizing to a target sequence of the host cell to guidethe Cas nuclease to the target sequence in the host cell, whereby thetarget sequence is modified by the HM-CRISPR/Cas system and the hostcell is killed or growth of the host cell is reduced, wherein themicrobiota comprises a second species of bacteria that is different fromthe species of the pathogenic host cells, and wherein growth of thesecond species of bacteria is not substantially reduced, therebyreducing the proportion of host cells in the microbiota; and wherein themicrobiota is a kidney microbiota or urethra microbiota.
 16. The methodof claim 15, wherein the HM-crRNA is comprised by a single guide RNA(sgRNA).
 17. The method of claim 15, wherein the engineered nucleic acidsequence is administered intravenously to the subject.
 18. The method ofclaim 15, wherein the engineered nucleic acid sequence is administeredby inhalation to the subject.
 19. The method of claim 15, wherein thenucleic acid sequence encoding the HM-crRNA is under a constitutivepromoter.
 20. The method of claim 15, wherein the Cas is a wild-typehost cell endogenous Cas3 and/or CASCADE Cas.
 21. The method of claim 1,wherein the engineered nucleic acid sequence is comprised by a vector,wherein the vector is a virus, phage, prophage or conjugative plasmid.22. The method of claim 15, wherein the Cas is a wild-type host cellendogenous Cas9.
 23. The method of claim 1, wherein growth of thepathogenic host cells is inhibited by at least 1000 fold.
 24. The methodof claim 15, wherein growth of the pathogenic host cells inhibited by atleast 1000 fold.